Review of HPLC Methods & HPLC-MS Detection For Direct Determination of Aspirin With Its Metabolites in Biological Matrices

Special issue: review
Received: 22 November 2011, Accepted: 5 December 2011 Published online in Wiley Online Library: 2 February 2012
(wileyonlinelibrary.com) DOI 10.1002/bmc.2694
Review of HPLC methods and HPLC methods with mass spectrometric detection for direct determination of aspirin with its metabolite(s) in various biological matrices
Ramesh Mullangia*, Kuldeep Sharmaa and Nuggehally R. Srinivasb
ABSTRACT: Aspirin, the most widely used drug in the world, has been known to mankind for over a century. It is not only the pharmacologically active entity, but is also biotransformed into a major metabolite, i.e. salicylic acid, which also exhibits similar pharmacologic/pharmacodynamic properties. Hence it is necessary to quantitate aspirin along with its metabolite(s) in various biological matrices accurately and precisely to correlate with pharmacological/pharmacodynamic activity. This paper provides a comprehensive overview of various bioanalytical methods (HPLC and LC-MS/MS) that have been reported for direct quantitation of aspirin along with its metabolite(s). The review also provides general information on sample collection, sample processing, internal standard selection, conditions for chromatographic separation, succinct validation data and applicable conclusions for reported assays in a structured manner. Copyright 2012 John Wiley & Sons, Ltd. Keywords: aspirin; metabolites; HPLC; LC-MS/MS; bioanalytical methods; review
Introduction
Aspirin (acetyl salicylic acid, ASA, Fig. 1, CAS no. 50-78-2) is an analgesic, anti-inammatory and antipyretic drug and the most widely used anionic drug in the world. In addition, low-dose aspirin is employed as an antithrombotic agent to inhibit cyclooxygenase-dependent platelet aggregation (Hennekens et al., 1989). After administration of aspirin, it is rapidly hydrolyzed in the body (by ubiquitous esterases in the gut wall, liver and other tissues) to produce salicylic acid (SA, Fig. 1, CAS no. 69-72-7), which is the principle metabolite and primarily responsible for the pharmacological activity of aspirin. The half-life of aspirin in plasma is about 20 min, suggesting that it is a short-lived entity (Reilly and FitzGerald, 1988; Kwong, 1987). However, the residence of SA is relatively longer in the circulation, with a half-life of 2.54.0 h. SA further undergoes direct conjugation with glycine to form salicyluric acid (SUA, Fig. 1) and with glucuronic acid to form acyl and phenyl glucuronide conjugates (Mason and Winer, 1981). Earlier work also reported that salicyluric acid forms a double conjugate with glucuronic acid (Hutt et al., 1982). To a minor extent, salicylic acid is metabolized by hydroxylation to gentisic acid (GA; 2,5-dihydroxybenzoic acid, Fig. 1), which itself is either conjugated with glycine to give gentisuric acid (Fig. 1, GUA) or is glucuronidated (Wilson et al., 1978).
bioanalytical strategies and considerations for method development to aid the LC-MS/MS analysis of aspirin and its metabolites. Kwong (1987) reviewed most of the reported HPLC methods for the quantication of aspirin along with its metabolites or metabolites alone. Hence the focus of our article is to provide a comprehensive review of both HPLC and LC-MS/MS methods and as well as discussing the advantages and limitations of these methods. In order to make it more benecial to the readership, we have provided a concise compilation of the validation details and applicability of the HPLC and LC-MS/MS methods in Table 1 and 2, respectively.
Sample matrix Blood or its derived products (serum and plasma) are typically the choice of sampling matrix for detecting and quantifying drugs in in vivo models. ASA is rapidly hydrolyzed by esterases in the gut wall, liver, plasma and red blood cells to form SA, with a half-life of only 20 min (Reilly and FitzGerald, 1988). Measurement of ASA in studying the metabolism of ASA is meaningful
* Correspondence to: Ramesh Mullangi, Drug Metabolism and Pharmacokinetics, Jubilant Biosys Ltd, Industrial Suburb, Yeshwanthpur, Bangalore-560 022, India. E-mail: mullangi_ramesh@jubilantinnovation.com
a
Scope
The purpose of this review is: (a) to enlist the various HPLC and LC-MS/MS methods in biological matrices for direct quantitation of aspirin alone or along with its metabolites, viz. salicylic acid, salicyluric acid and gentisic acid, with other relevant information such as sample processing details, chromatographic conditions, validation parameters in tabular format; and (b) discuss relevant
Drug Metabolism and Pharmacokinetics, Jubilant Biosys Ltd, Industrial Suburb, Yeshwanthpur, Bangalore-560 022, India Vanthys Pharmaceutical Development (Pvt) Ltd, Phoenix Pinnacle, Ulsoor Road, Bangalore-560 001, India Abbreviations used: ASA, acetyl salicylic acid; GA, gentisic acid; GUA, gentisuric acid; LLE, liquidliquid extraction; PPT, protein precipitation; SA, salicylic acid; SPE, solid-phase extraction; SUA, salicyluric acid.
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Copyright 2012 John Wiley & Sons, Ltd.
Review of aspirin bioanalytical methods

O OH O OH O CH3 Hydrolysis by esterases O OH OH conjugation with glycine O NH OH
Aspirin or acetyl salicylic acid (ASA)

Hydroxylation
Salicylic acid (SA)
Salicyluric acid (SUA)
OH OH
conjugation with glucuronic acid
Acyl and phenyl glucuronides of salicylic acid
HO
conjugation with glycine
O OH O NH OH
Gentisic acid (GA)
conjugation with glucuronic acid
Gentisic acid glucuronide
HO
Gentisuric acid (GUA)
Figure 1. Metabolic pathway of aspirin.
only if in vitro hydrolysis of ASA to SA is inhibited/arrested. Therefore blood specimens should be collected in chilled tubes containing esterase inhibitor. Nieder and Jaeger (1983) published the results of extensive studies on ASA hydrolysis in biological matrices and concluded that enzymatic hydrolysis of ASA is drastically reduced by potassium uoride; with additional cooling, the loss of ASA was less than 1% in 15 min in plasma. Further, no detectable loss was seen in 5 days for frozen plasma samples (at 1 C) while only a 510% loss was observed after 4 weeks of storage. Interestingly, McMahon and Kelly (1998) published their observations, in which the acidication of plasma samples appeared to be a more important driver than temperature in terms of the stability of ASA. Numerous methods have been reported for quantitation of ASA and its metabolites (mostly deacetylated product, i.e. salicylic acid) in plasma, while only one has been reported for serum (Wahlin-Boll et al., 1981). Several methods have been reported for urine as the choice of matrix for analysis (Amick and Mason, 1979; Harrison et al., 1980; Bakar and Niazi, 1983; Mays et al., 1984; Krivoskov et al., 1996). However as reported by Krivoskov et al. (1996), ASA is eliminated from systemic circulation quickly but not excreted in urine, which unequivocally points to its fast deacetylation in kidney, as the drug is ltered in glomeruli into primary urine. SA is a dominant plasma metabolite and also excreted in urine (40% of administered dose). In addition, some authors have also reported methods in tissue homogenates (Reidl, 1983) and skin (Pirola et al., 1998). Sample preparation Efcient sample preparation is key requirement for a good analytical method that leads to improved selectivity, sensitivity and ruggedness. Removal of interfering matrix compounds/
components is required to lower or eliminate the risk of matrix effects in LC-MS procedures. During the pre-treatment, endogenous components such as proteins, salts and lipids are removed from biological matrix. These components can inuence the ionization efciency of MS and therefore can modify the sensitivity of method. The most widely used techniques for sample preparation are liquidliquid extraction (LLE), solid-phase extraction (SPE) and protein precipitation (PPT). Protein precipitation. Almost all methods reported for quantication of ASA, SA and other metabolites include the addition of acid to sample matrix to bring down the sample matrix pH close to the pKa of ASA (i.e. 3.5). The earliest method reported by Cham et al. (1980) included addition of an equal volume of methyl cyanide (acetonitrile) and analysis of supernatant layer for estimation of ASA and SA in human plasma. In 1981, Rumble et al. reported a precipitation method with methanol, which was developed with the advantages of lower injection volume and addition of an internal standard (IS) in the methanolic solution. Further, several other methods with acetonitrile (Bakar and Niazi, 1983; Kees et al., 1996) and perchloric acid (OKruk et al., 1984; Krivoskov et al., 1996) were reported. All of these methods were developed for UV detection (235280 nm). Recently in 2009, Xu et al. reported their method utilizing acidied acetonitrile as precipitation solution and mass spectrometric detection. The method was validated for the intended usage, although the matrix effect and ion suppression were evaluated for ASA and SA and slight ion suppression was seen from the results reported (i.e. recoveries for ASA concentrations of 8 and 400 ng/mL were 51 and 84%, while those for SA concentrations of 80 and 4000 ng/mL recoveries were 102 and 60%). From the literature data it is apparent that the precipitation method should not be the primary choice for processing such complex analytes.
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Sample processing details Matrix: rat plasma (100 mL). System: HPLC with UV detector. Linearity: 6020,000 ng/mL for aspirin and salicylic acid and 20020,000 ng/mL for 2,3-DHBA and gentisic acid (r2 > 0.999 for all analytes). Selectivity: no endogenous interference was observed from lasma samples at the retention times of the analytes. Chromatographic conditions Validation parameters Applicable conclusions Extraction: to an aliquot of plasma equal volume of 0.6% phosphoric acid aqueous solution was added and centrifuged for 10 min at 1000 rpm and the contents were loaded on SPE column for on-line sample extraction and enrichment. Following this the enriched samples were analyzed on an analytical column. Internal standard: no IS was used. Mobile phase: gradient elution with mobile phase A and B. Mobile phase A is a mixture of waterACN (1000:10, v/v) containing 0.2% TFA and mobile phase B is a mixture of waterACN (100:900, v/v) containing 0.1% TFA. Initially mobile phase B was 0%, which was increased to 30% by 18 min, after which the concentration was held at 30% for 2 min. After each run both SPE and analytical column were ushed with mobile phase B. Flow-rate for SPE and analytical column was 2.8 and 1 mL/min, respectively. Detection: lmax set at 235 nm. Volume of injection: 10 mL. Accuracy and precision: intraand inter-day precision were 0.15.8% and 0.211.4%, respectively, for all analytes. For 2,3-DHBA and gentisic acid, precision and accuracy could not be determined below 60 ng/mL because of interference from plasma samples. The intra- and interday accuracy were 92.6112% and 98.2103.5%, respectively. Column: for LC-UV analysis: MCSAX (10 4 mm, 50 mm, 12 nm pore size) maintained at 35 C; whereas for SPE-LCUV analysis: MC-SAX-SPE was used as extraction column and YMC Hydrosphere C18 (150 4.6 mm, 5 mm, 12 nm pore size) was used as an analytical column. Authors discussed the restricted access media (RAM) columns [made up of methyl cellulose (MC) immobilized silica materials bonded to octadecyl silanized (ODS) silica] advantages for separation of broad spectrum of drugs (cationic, anionic, and neutral). MC-strong anionexchange (MC-SAX) has trimethyl ammonium groups on pore surface of MC silica, used for anionic analytes on-line extraction from biosamples. Various mobile phase combinations and inuence of organic solvent on compound recovery over MC-SAX-SPE column were evaluated. This method was successfully applied to quantitate aspirin, salicylic acid, 2,3-DHBA and gentisic acid following i.v. administration of aspirin to rats at 2.5 mg/kg dose. In the plasma samples 2,3-DHBA and gentisic acid could not be determined, hence pharmacokinetic (PK) proles of aspirin and salicylic acid were established and these results were in agreement with earlier reported values. R. Mullangi et al.
Table 1. Summary validation of various published HPLC methods
Analyte(s)
Authors
Aspirin, salicylic acid, 2,3DHBA and gentisic acid
Yamamoto et al., 2007
Table 1. (Continued) Sample processing details Retention time: ~13.5, 15, 18.5 and 21 min for gentisic acid, 2,3-DHBA, aspirin and salicylic acid, respectively. Total run time: 23 min. System: HPLC with UV detector. Linearity: 1001000 ng/mL. Limit of detection: 200 ng/mL. Column: mBondapak C18 (300 3.9 mm, 10 mm) coupled to a guard column (Supelco, 200 4 mm, 5 mm) maintained at ambient room temperature. This method was used to quantitate aspirin individually or in combination with pyridostigmine bromide, caffeine and acetaminophen in rat plasma, urine and tissues in a PK study. Chromatographic conditions Validation parameters Applicable conclusions
Analyte(s)
Authors
909
Aspirin
Abu-Qare and Abou-Donia, 2001
Matrix: rat plasma and urine (200 mL). Extraction: an aliquot of plasma/urine was acidied with 1 M acetic acid (pH 5), vortex mixed and centrifuged for 5 min at 1000 g and the supernatant was loaded on C18 Sep Pak Vac 3 cc, 500 mg (preconditioned with 3 mL ACN and 3 mL water), washed with 2 mL water and nally eluted twice with 2 mL MeOH. The combined eluate was concentrated to 500 mL under gentle stream of nitrogen and transferred into HPLC vials for analysis. Internal standard: no IS was used. Mobile phase: gradient elution at a ow-rate of 1 mL/min. Detection: lmax set at 280 nm. Volume of injection: 10 mL. Retention time: 8.8, 9.9, 10.4 and 11.5 for acetaminophen, pyridostigmine bromide, caffeine and aspirin, respectively. Total run time: 15 min. Selectivity: no endogenous interference from plasma and urine at the retention times of the analytes. Absolute recovery: 88.6 9.3 and 85.9 9.8% in plasma and urine, respectively. Accuracy and precision: precision and accuracy was found to be acceptable.
(Continues)
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Sample processing details Matrix: human plasma (200 mL). System: HPLC with UV detector. Linearity: 0.15 mg/mL for aspirin and and 0.2515 mg/mL for salicylic acid (r > 0.999 for both analytes). Limit of detection: 0.04 mg/mL for both the analytes. Chromatographic conditions Validation parameters Applicable conclusions Extraction: to an aliquot of chilled plasma equal volume of 0.2 M orthophosphoric acid was added, vortex mixed and centrifuged at 5800 g for 3 min (to remove the cloudiness, if any) and 200 mL was loaded over on-line SPE column (PEEK cartridge having Hypersil C18, 30 mm) and eluted with water ortho-phosphoric acid (1000:1, v/v, pH 2.5). Internal standard: no IS was used. Mobile phase: isocratic mobile phase comprising waterMeOHACNorthophosphoric acid (650: 200:150:1, v/v/v/v, nal pH 2.6) at a ow-rate of 1 mL/min. Column: Nucleosil C8 (250 4.6 mm, 5 mm) coupled to a Hypersil C8 guard column (10 4 mm, 10 mm) maintained at ambient room temperature. Authors evaluated various anticoagulants (lithium heparin, EDTA, uoride/EDTA and citrate) to be used during blood collection and found that minimal difference in plasma proles. Fluoride/EDTA was the choice of anti-coagulant as it gave cleaner baseline chromatograms with blank plasma and inhibits the action of plasma esterases, which catalyzes in vitro hydrolysis of aspirin. By conducting a series of experiments the authors recommended that acidication of plasma samples is more important than maintaining them in chilled condition to minimize the hydrolysis of aspirin. This columnswitching method was used for the quantication of aspirin and salicylic acid following oral administration of 600 mg of aspirin to healthy human volunteer under fed conditions. Detection: lmax set at 225 nm. Selectivity: no endogenous interference at the retention times of the analytes evaluated from six different sources. A number of possible commonly coadministered drugs (19 drugs) were evaluated and found that only xylazine and prazosin interfere with the chromatographic resolution of aspirin and salicylic acid. Recovery: absolute recovery at 0.5 and 5 mg/mL was found to be 99 and 100% for aspirin; 104 and 101% for salicylic acid. The relative recovery at 0.5 and 5 mg/mL was 101 and 94% for aspirin; 88 and 90% for salicylic acid. R. Mullangi et al.
Table 1. (Continued)
Analyte(s)
Authors
Aspirin and salicylic acid
McMahon and Kelly, 1998
Table 1. (Continued) Sample processing details Volume of injection: Not applicable Chromatographic conditions Validation parameters Applicable conclusions
Analyte(s)
Authors
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Retention time: 11.5 and 15.6 min for aspirin and salicylic acid, respectively. Total run time: 22 min.
Pirola et al., 1998
Matrix: human skin and plasma.
System: HPLC with UV detector.
Column: LiChrospher 100 RP18 (250 4 mm, 5 mm) coupled to a 2 cm pre-column (lled with analytical column material) maintained at ambient room temperature.
Accuracy and precision: intraand inter-assay precision and accuracy found to be acceptable for both aspirin and salicylic acid. Stability: aspirin was found to be stable for 24 h at room temperature, at 4 C for up to 48 h and at 30 C and through 2 F/T cycles. After these time periods the levels of endogenous interference increased, which adversely affected the quantication of both analytes. Linearity: 0.1100 and 0.15 mg/cm2 for aspirin and salicylic acid in tape strippings; 0.12 and 0.150 mg/mL for aspirin and salicylic acid in plasma (r2 > 0.9997 for both analytes). Selectivity: no endogenous interference at the retention times of the analytes and corresponding internal standards. No aspirin or salicylic acid was detected in plasma following topical application of 750 mg aspirin. Following oral administration of 500 mg of aspirin both aspirin and salicylic acid were quantied using this method and the values were close to the earlier reported values.
Extraction: to the tape strippings 3 mL of ACN was added, sonicated for 15 min and centrifuged for 10 min at 15000 g. The aliquot was used for HPLC analysis. To an aliquot of plasma (1 mL), IS solution (200 mg) and 1 mL of 2 M HCl were added, vortex mixed for 1 min and centrifuged for 10 min at 1500 g and the solution was extracted overt SPE column (Isolute C8, pre-washed with 2 vols of MeOH and 1 vol. of 0.1 M HCl) and washed with 5 vols Mobile phase: isocratic mobile phase comprising waterphosphate buffer (pH 2.5)ACN, 35:40:25 (v/v/v) at a ow-rate of 1 mL/min.
Absolute recovery: >98% for aspirin, salicylic acid in both the matrices.
(Continues)
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Sample processing details Chromatographic conditions Validation parameters Applicable conclusions of 2 M HCl and the SPE was kept in dried state for 15 min. After 15 min, SPE was nally eluted with 2 500 mL of MeOH1% NH4OH ACN, 50:30:20 (v/v/v). Combined eluates were used for HPLC analysis. Internal standard: piroxicam (for tape strippings) and phenobarbital (for plasma). Detection: lmax set at 234 nm Volume of injection: 50100 mL. Accuracy and precision: in tape strippings, mean intra-day accuracy was 98.8 and 99.3% with a precision of 2.3 and 1.8% for aspirin and salicylic acid, respectively. Similarly the inter-day accuracy was 100.2 and 98.8% with a precision of 2.8 and 1.5% for aspirin and salicylic acid, respectively. In plasma, mean intra-day accuracy was 101.3 and 99.7% with a precision of 4.3 and 3.3% for aspirin and salicylic acid, respectively. Similarly the inter-day accuracy was 98.1 and 99.5% with a precision of 3.8 and 1.7% for aspirin and salicylic acid, respectively. Stability: aspirin and salicylic acid were found to be stable in both matrices for one month at 25 C. R. Mullangi et al. Matrix: human plasma (200 mL). Retention time: 6.8, 8.7, 16.0 and 21.6 min for aspirin, salicylic acid, phenobarbital and piroxicam, respectively. Total run time: ~25 and 30 min for plasma and tape strippings, respectively. System: HPLC with UV detector. Linearity: 0.220 mg/mL for aspirin and and 0.550 mg/mL for salicylic acid (r2 > 0.9997 for both analytes). This method was used for the analysis of human plasma samples followed by administration of 100500 mg of aspirin to healthy
Analyte(s)
Authors
Kees et al., 1996
Table 1. (Continued) Sample processing details Column: Novapak C18 (10 4 mm, 4 mm) maintained at 35 C. Limit of detection: 75 and 100 pg on column for aspirin and salicylic acid, respectively. human volunteers in a bioequivalence study. Chromatographic conditions Validation parameters Applicable conclusions Review of aspirin bioanalytical methods
Analyte(s)
Authors
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Extraction: to an aliquot of plasma equal volume of IS solution, which brings the pH of mixture to 2.7. To this, 400 mL of ACN was added mixed and after 15 min, the contents were centrifuged at 10,500 g for 1 min and the supernatant was transferred into 1.5 mL tubes containing 100 200 mg sodium chloride. The suspension was vortex mixed and incubated at 4 C for 10 min. Following incubation the contents were centrifuged for 1 min at 10,500 g and 200 mL of organic layer was transferred into HPLC vials for analysis. Internal standard: 2methyl benzoic acid (5 mg/mL in 1:1 mixture of 0.2 M HCl and 0.2 M ortho-phosphoric acid). Mobile phase: isocratic mobile phase comprising 740 mL water, 900 mL ortho-phosphoric acid and 180 mL ACN (nal pH 2.5) at a ow-rate of 1 mL/min. Detection: lmax set at 237 nm. Selectivity: no endogenous interference at the retention times of the analytes.
Volume of injection: 10 mL.
Retention time: 2.1, 2.9, 4.2, 6.8 and 8.9 min for gentisic acid, salicyluric acid, aspirin,
Absolute recovery: 106.8 8.4, 121.7 4.8 and 129.0 2.1% for aspirin, salicylic acid and IS, respectively. Accuracy and precision: precision and accuracy determined at 100 ng/mL for aspirin and salicylic acid and found to be acceptable. Stability: aspirin was found to be stable in human plasma for 3 months at 70 C, but at 30 C it decomposed to (Continues)
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Sample processing details salicylic acid and IS, respectively. Total run time: 10 min. Chromatographic conditions Validation parameters Applicable conclusions Matrix: human plasma and urine (200 mL). Extraction: an aliquot of plasma was deproteinized with 50 mL of 35% perchloric acid, vortex mixed for 10 s and centrifuged at 12,000 g for 10 min and 50 mL supernatant was transferred into HPLC vials for analysis. Internal standard: no IS was used. Mobile phase: isocratic mobile phase comprising water85% phosphoric acidbutanol tetrabutylammonium hydroxideMeOH (134:1:1:63, v/v/v/v) at a ow-rate of 0.9 mL/min. Detection: lmax set at 237 and 305 nm for plasma and urine samples, respectively. Retention time: 2.86, 3.78 and 7.12 min for aspirin, salicylic acid and salicyluric acid, respectively. Total run time: 10 min. System: HPLC with UV detector. Column: Separon SGX C18 (150 3.3 mm) maintained at 45 C. System: HPLC with UV detector. salicylic acid within 3 weeks to 5% and 7 weeks to 13%. At 24 C aspirin degradation was 5% within 1 h and addition of potassium uoride has no inuence. Linearity: 0.210 mmol/L for all analytes (r2 > 0.999 for both analytes). Absolute recovery: were 90105% for all analytes. Blood samples (1 mL) were collected into tubes containing heparin (50 IU) and sodium uoride (4 mg per 1.5 mL blood) and immediately centrifuged to harvest plasma. Plasma was stored at 56 C until analysis (within one week). The method was used to quantitate aspirin, salicylic acid and salicyluric acid following oral administration of 30 mg aspirin and also to quantitate salicylic acid and salicyluric acid following oral administration of 30400 mg of aspirin. Matrix: rabbit whole blood, plasma and isolated erythrocytes. Linearity: 20400, 100500 and 240 mg/mL for aspirin, salicylic acid and gentisic acid, respectively (r > 0.98 for all analytes in three matrices except for gentisic acid in To the heparinized rabbit blood potassium uoride (50 mL/5 mL) was added to prevent hydrolysis of aspirin by cholinesterases. Plasma and erythrocytes (by spinning plasma at 1500 g for 10 min) were harvested from blood. Whole R. Mullangi et al.
Analyte(s)
Authors
Aspirin, salicylic acid and salicyluric acid
Krivoskov et al., 1996
Aspirin, salicylic acid and gentisic acid
Klimes et al., 1992
Table 1. (Continued) Sample processing details Chromatographic conditions Validation parameters Applicable conclusions
Analyte(s)
Authors
915
Column: Separon SGX C18 (150 3.2 mm, 5 mm) maintained at ambient room temperature.
plasma, wherein it was r > 0.89). Limit of detection: 20 ng/mL for aspirin and salicylic acid in blood and plasma; 60 ng/mL in erythrocytes. For gentisic acid it was 50 ng/mL in blood and plasma.
blood, erythrocytes and plasma were frozen until analysis. This method was used to quantitate (up to 2 h) aspirin, salicylic acid and gentisic acid in rabbit whole blood, plasma and erythrocytes following i.v. administration of aspirin (Aspegic injection) to rabbits at 100 mg/kg dose. Following i.v. administration at 3 min, aspirin evenly distributed between erythrocytes and plasma and its levels decreased rapidly in both matrices. Gentisic acid was not detected in erythroctyes in the course of 2 h PK study time.
Mobile phase: isocratic mobile phase comprising MeOH water, 80:100, v/v (nal pH 2.5 with 5% perchloric acid) at a ow-rate of 0.5 mL/min.
Selectivity: no endogenous interference at the retention times of the analytes in all the matrices.
Extraction: blood samples: to an aliquot of blood (500 mL), 10 mL IS solution was added and the blood was hemolyzed by adding 900 mL of water. The contents were shaken for 5 min, sonicated for 5 min and left aside for 5 min at room temperature. Then the sample was acidied with 300 mL of HCl (3 mol/L), shaken for 5 min and 6 mL of dichloromethane was added, centrifuged for 5 min at 1930 g. The organic layer (5 mL) was evaporated to dryness under nitrogen stream and residue was reconstituted in 50 mL of mobile phase and used for HPLC analysis. Erythrocytes: 500 mL was processed in the same manner as described for blood samples except using 400 mL of HCl instead of 300 mL for acidication and centrifugation was 7 min. Plasma: 500 mL was processed in the same manner as described for blood samples except Detection: lmax set at 236 nm. Absolute recovery: mean recovery was 98.9 1.8, 90.7 4.2 and 100 1.6% for aspirin from whole blood,
(Continues)
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Sample processing details using 200 mL of HCl instead of 300 mL for acidication. Chromatographic conditions Validation parameters Applicable conclusions Internal standard: benzanilide. erythrocytes and plasma, respectively; 70.9 1.5 and 61.7 3.8, 80.1 1.4% for salicylic acid from whole blood, erythrocytes and plasma, respectively; 50.3 3.7 and 67.2 3.3% for gentisic acid from whole blood and plasma, respectively. Precision and accuracy: intra- and inter-day precision and accuracy were found to be within acceptable limits (with CV% <5). Matrix: human plasma (500 mL). Retention time: 3.0, 5.3, 8.4 and 14.2 min for gentisic acid, aspirin, salicylic acid and IS, respectively. Salicyluric acid eluted at 4 min had endogenous interference. Volume of injection: 10 mL. Total run time: 20 min. System: HPLC with UV detector. Extraction: to an aliquot of plasma, 0.5 mL 1 M oxalic acid and 0.5 mL, 25 mM phosphate buffer (H3PO4: KH2PO4, pH 2.5) containing IS were added, vortex mixed with 5 mL of ether: hexane (1:1, v/v) for 2 min and centrifuged for 3 min at 3000 rpm. The organic phase was transferred into another tube containing 200 mL of 0.5 M phosphate buffer (KH2PO4 Na2HPO4, pH 7) and the contents were vortex Column: Bondapak phenyl (300 4.6 mm, 10 mm) maintained at ambient room temperature. Linearity: 0.05200 mg/mL for aspirin and salicyluric acid; 0.1200 mg/mL for salicylic acid and 0.5200 mg/mL for gentisic acid. Selectivity: no endogenous interference at the retention times of the analytes. Interference by other 15 drugs was tested and found that they did not interfere with the assay. It was noticed that back-extraction process avoided the normal basic drug interference. Authors evaluated the effect of TEA concentration in mobile phase on resolution of analytes and its role in decreasing the endogenous interference. Back-extraction was adopted to minimize the loss of aspirin and salicylic acid by sublimation during sample processing steps. The validated ion-pair HPLC method was applied to analyze plasma samples collected from healthy human volunteer and pre-eclamptic woman following oral administration of 100 mg of slowrelease aspirin formulation. R. Mullangi et al.
Analyte(s)
Authors
Aspirin, salicylic acid, gentisic acid and salicyluric acid
Shen et al., 1990
Table 1. (Continued) Sample processing details Chromatographic conditions Validation parameters Applicable conclusions
Analyte(s)
Authors
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mixed for 2 min and centrifuged for 3 min. An aliquot of 100 mL of aqueous portion was mixed with 50 mL of 25% H3PO4 and a 20 mL aliquot was used for HPLC analysis. Internal standard: m-hydroxybenzoic acid (50 mg/mL). Absolute recovery: 92100% for all the analytes.
Siebert and Bochner, 1987
Matrix: human plasma (1 mL).
Mobile phase: isocratic mobile phase comprising MeOH waterTEA, 280:717.5: 2.5, v/ v/v nal pH 3.5) at a ow-rate of 1 mL/min. Detection: lmax set at 229 nm. Retention time: 5.5, 8.6, 10.1 and11.6 min for gentisic acid, salicyluric acid, salicylic acid and aspirin, respectively. Total run time: 12 min. System: HPLC with uorescence detector.
Extraction: to an aliquot of plasma, 3 mL of diethyl ether, 50 mL of 21.25% phosphoric acid (85% ortho-phosphoric acidwater, 1:3, v/v) were added vortex mixed for 10 min and centrifuged for 5 min at 1500 g. To the ether phase150 mL of 10 mM phosphate buffer (pH 7.4) was added, vortex mixed and centrifuged. The ether layer was evaporated under nitrogen stream and left over aqueous layer was kept on ice until HPLC analysis. Column: Spherisorb ODS 2 (50 4.6 mm, 5 mm) maintained at ambient room temperature. Mobile phase: isocratic mobile phase comprising 40% MeOH with 0.085% phosphoric acid in water at a ow-rate of 1 mL/min. Sodium hydroxide (0.5 M) at 0.15 mL/min was merged via T-piece with the eluent from the column, which is owed through reaction coil (16 m 0.25 mm, i.d. with coil diameter of 6 cm), which is immersed in an oil batch maintained at
Linearity: 2 ng/mL to 2 mg/mL for aspirin and 20 ng/mL to 50 mg/mL for salicylic acid (r2 > 0.999 for both analytes). Selectivity: no endogenous interference at the retention times of the analytes. No interfering peaks were found in extracts of plasma from patients taking medications (almost 30 drugs). Absolute recovery: 74.6 4.4 and 70.2 3.8% for aspirin in 0.5 and 1 mL plasma, respectively; 77.0 2.4 and 66.4 1.6% for salicylic acid in 0.5 and 1 mL plasma, respectively.
Owing to post-column hydrolysis and uorescence detection, aspirin can be detectable as low as 2 ng/mL in plasma. Authors optimized the pH, temperature of the coil and time duration to control the rate of aspirin hydrolysis. This method is used to quantitate aspirin and salicylic acid following oral administration of 50 mg of aspirin to healthy human volunteers.
(Continues)
918
Sample processing details Internal standard: no IS was used. Chromatographic conditions Validation parameters Applicable conclusions Matrix: human plasma (200 mL). Column: Lichrosorb RP8 (250 4 mm, 7 mm) maintained at ambient room temperature. 60 C. The separated and hydrolyzed aspirin was measured as salicylic acid by uorescence detector with an excitation wavelength of 310 nm and a 389 nm emission. Retention time: ~5.0, 7.0 and 12.0 min for salicyluric acid, aspirin and salicylic acid, respectively. Volume of injection: 50 mL. Total run time: 12 min. System: HPLC with UV detector. Accuracy and precision: found to be within the acceptable limits. Linearity: 0.120 mg/mL for aspirin and salicylic acid (r2 > 0.999 for both analytes). Selectivity: no endogenous interference at the retention times of the analytes. Extraction: to an aliquot of plasma, 15 mL of 7 M H3PO4, 80 mg NaCl and 40 mL waterMeOH (1:1, v/v) and 50 mL IS solution were added, vortex mixed for 15 s, then 8 mL of hexane was added and shaken for 10 min at 0 C. The organic layer was dried under nitrogen stream and the residue was reconstituted in 200 mL mobile phase and was used for HPLC analysis. Internal standard: p-toluic acid (20 mg/mL in ACNwater, 70:30, v/v). Mobile phase: isocratic mobile phase comprising ACNwater (pH 2.5 with H3PO4), 30:70, v/v at a ow-rate of 1 mL/min. Detection: lmax set at 229 nm. Retention time: 5.9, 8.0 and 9.6 min for aspirin, salicylic acid and IS, respectively. Volume of injection: 100 mL. Total run time: 12 min. Absolute recovery: 27 3 and 54 2% for aspirin and salicylic acid, respectively. Accuracy and precision: found to be within the acceptable limits. Blood samples (1 mL) were collected into chilled tubes containing 10 mL heparin (1000 U/mL) and potassium uoride (10 mL 50% w/v in water). Plasma was harvested immediately by spinning at 0 C and stored at 80 C until analysis (within one week). Although dichloromethane gave recovery >90% for aspirin in healthy volunteers, rats and rabbits, the recovery was poor from uremic patients plasma. Only hexane eliminated interference and gave good recovery of aspirin from uremic patients plasma. No loss of salicylic acid was observed during evaporation step. The validity of the method was determined by assessing the plasma concentrations of aspirin and salicylic acid following i.v. administration of aspirin at a dose of 100 mg/m2 to a uremic patient, who is on regular hemodialysis. R. Mullangi et al.
Analyte(s)
Authors
Gaspari and Locatelli, 1987
Table 1. (Continued) Sample processing details Matrix: human serum (500 mL). Column: Spherisorb ODS (250 4.5 mm, 5 mm) maintained at ambient room temperature. System: HPLC with UV detector. Linearity: 10120 mg/mL for both the analytes (r2 > 0.998 for both analytes). Authors suggested that this method can be routinely used in hospitals to quantitate aspirin and salicylic acid along with paracetamol. Chromatographic conditions Validation parameters Applicable conclusions
Analyte(s)
Authors
919
Tebbett et al., 1985
Extraction: an aliquot of serum was acidied with 100 mL of HCl and extracted thrice with 2 mL of chloroform ACN (60:40, v/v). The combined organic extracts were evaporated to dryness and reconstituted in 100 mL of mobile phase and was used for HPLC analysis. Internal standard: no IS was used. Mobile phase: isocratic mobile phase comprising ACN MeOHwater, 25:10: 65 ( v/v/v) nal pH 3.0 with ortho-phosphoric acid at a ow-rate of 1 mL/min. Detection: lmax set at 234 nm. Limit of detection: 2 ng on column for both analytes.
Brandon et al., 1985
Retention time: ~3.5 and 5.0 min for aspirin, and salicylic acid, respectively. Volume of injection: 20 mL. Total run time: 9 min. System: HPLC with UV detector.
Selectivity: no endogenous interference from the blank serum at the retention times of the analytes. Absolute recovery: 97100% for both analytes.
Extraction: to an aliquot of plasma, 200 mL of 1 M
Column: m Bondapak C18 (300 3.9 mm, 10 mm) coupled to a
Linearity: 0.0250.5 and 0.55 mg/mL as low concentration curve and 0.510 and 570 mg/mL as high concentration curve for aspirin and salicyluric acid, respectively (r2 > 0.998 for both analytes). Selectivity: no endogenous interference at the retention
Peroxide-free diethyl ether or anhydrous diethyl ether (used after 72 h) showed chromatographic interference with aspirin. To prevent the enzymatic hydrolysis of aspirin into salicylic acid, physostigmine sulfate was added to blood samples. This method was used to determine the PK parameters of aspirin and salicylic (Continues)
920
Sample processing details HCl and 10 mL of anhydrous diethyl ether were added and the tubes were closed, vortex mixed for 5 min and centrifuged at 1500 g for 4 min. The organic layer was dried under gentle stream of nitrogen while keeping the tubes in an ice water bath (to prevent sublimation of salicylic acid). Residue was dissolved in 200 mL of mobile phase and used for HPLC analysis. IS solution was added into tubes and dried before adding plasma. Internal standard: m-anisic acid (25 mL of 50 mg/L in MeOH). times of the analytes. Interference from other 46 drugs and from plasma of patients receiving other medications was tested and found that there was no interference except with methyclothiazide. Mobile phase: isocratic mobile phase comprising 0.13 mL ortho-phosphoric acid, 10 mL 1-butanol, 270 mL MeOH and 720 mL water delivered at a ow-rate of 1.8 mL/min. Detection: lmax set at 234 nm. Retention time: 5.6, 8.0 and 9.6 min for aspirin, salicylic acid and IS, respectively. Volume of injection: 5100 mL. Total run time: 10 min. System: HPLC with UV detector. Absolute recovery: 96.4100.3 and 76.791.2%, respectively, for aspirin and salicylic acid, respectively. guard column (23 3.9 mm, m Bondapak C18 material) maintained at 47 C. Chromatographic conditions Validation parameters Applicable conclusions acid following oral administration of either 100 or 600 mg of glycinated aspirin to overnight fasted healthy human volunteers. Blood was collected into tube having lithium heparin and physostigmine sulfate (100 mL of 2 104 M). Blood samples were centrifuged within 5 min and stored at 20 C until analysis. The Cmax and Tmax observed for aspirin were 1.7 0.47 mg/mL and 26.7 10.8 min, respectively with a t of 14.7 min; for salicylic acid the Cmax and Tmax were 6.63 1.42 mg/mL, respectively and 1.67 0.88 h with a t of 1.61 h. All the obtained values were in agreement with the earlier reported values. Matrix: human and rat plasma or serum (200 mL) and rat urine. Linearity: 1500 mg/mL for aspirin and salicylic acid; 160 mg/mL for gentisic acid and salicyluric acid in plasma (r > 0.993 for all analytes). In rat urine the linearity range for salicylic acid was 1100 mg/mL (r > 0.996). Blood samples were collected into chilled tubes containing 5 mg/mL potassium uoride (25%) to prevent aspirin hydrolysis. Similarly following collection of urine samples, they were mixed with equal volume of 10 M hydrochloric acid and stored at 80 C until R. Mullangi et al.
Analyte(s)
Authors
OKruk et al., 1984
Table 1. (Continued) Sample processing details Extraction: plasma/serum samples, to an aliquot of plasma or serum 20 mL of 30% perchloric acid containing IS solution (0.02%) and 200 mL of MeOH were added vortex mixed and centrifuged at 9000 g for 4 min. Following centrifugation the supernatant was transferred into HPLC vials for analysis. Column: C8 (10 4 mm, 4 mm) coupled to a guard column (40 2 mm, 3038 mm) maintained at ambient room temperature. Chromatographic conditions Validation parameters Applicable conclusions Review of aspirin bioanalytical methods
Analyte(s)
Authors
921
Urine samples, after thawing urine samples, 400 mL of urine sample was transferred into 2 mL glass ampoules ushed with nitrogen and sealed immediately. The ampoules were heated for 3 h at 120 C to hydrolyze the conjugates. Following cooling, to the 20 mL of hydrolyzed mixture, 20 mL of IS solution (0.02%), 180 mL of distilled water and 200 mL MeOH were added; the contents were centrifuged at 9000 g for 2 min and 20 mL of supernatant was used for HPLC analysis. Internal standard: 3,4,5-trimethoxybenzaldehyde. Mobile phase: isocratic mobile phase comprising MeOH 0.1% KH2PO4 buffer (pH 3.9), 35: 65 (v/v) at a ow-rate of 2 mL/min. Detection: lmax set at 235 nm for plasma/serum analysis, whereas for urine samples the lmax was set at 313 nm.
Selectivity: no endogenous interference at the retention times of the analytes in plasma/serum. Nearly 19 benzoic acid derivatives were tested for their interference on the chromatographic resolution (in serum/plasma) of these analytes and found that all of them eluted before the rst analyte (i.e. gentisic acid) under this chromatographic conditions. In rat urine also there was interference at the retention time of salicylic acid and IS. Recovery: found to be 8086% for the analytes in serum/plasma. Accuracy and precision: precision and accuracy determined at 100 ng/mL (for aspirin and salicylic acid) and found to be acceptable. further analysis. Anticoagulant EDTA was preferred over heparin as benzyl alcohol, which is used as a preservative and interferes with the retention time of salicyluric acid. Authors evaluated the effect of MeOH concentration in mobile phase and different pH conditions on the chromatographic resolution of the analytes. This method was successfully used to study the PK of aspirin and its metabolites in rats and human. Rats were given i.v. infusion at a dose of 200 mg/kg and serum was analyzed for aspirin and its metabolites. In rat serum aspirin, salicylic acid and gentisic acid were detectable but salicyluric acid was not detectable. The serum half-life of aspirin was 78 min, which is less than plasma half-life (15 min) in man. Following oral administration of aspirin tablets (4 325 mg), plasma was analyzed and time vs concentration proles of aspirin, salicylic acid and salicyluric acid were determined, whereas gentisic acid was quantiable only at 0.5 and 4 h only.
(Continues)
922
Sample processing details Chromatographic conditions Validation parameters Applicable conclusions Matrix: plasma and urine of rabbits and humans. Volume of injection: 20 mL. Retention time: 3.3, 4.1, 5.5, 6.7 and 8.7 min for gentisic acid, salicyluric acid, aspirin, salicylic acid and IS, respectively, in plasma/ serum. In rat urine the retention time for salicylic acid and IS was 6.0 and 7.5 min, respectively. Total run time: 10 min. System: HPLC with UV detector. Extraction: plasma samples, to an aliquot of human plasma (1 mL), 150 mL of 3 M orthophosphoric acid, 400 mg of sodium chloride and 12 mL DCM containing IS (3 mg/mL) were added. The contents in tube were shaken for 10 min at 300 rpm and centrifuged. The upper layer was discarded and the organic layer (8 mL) was dried under reduced pressure and the residue was dissolved in 200 mL of mobile phase and used for HPLC analysis. In case of rabbit plasma Column: Nucleosil C18 (250 4.6 mm, 5 mm) maintained at ambient room temperature. Linearity: 0.2100 mg/mL for aspirin and salicylic acid; 0.24 mg/mL for salicyluric acid in plasma. In urine: 220, 20300 and 2002000 mg/mL for gentisic acid, salicylic acid and salicyluric acid, respectively (r2 > 0.999 for all analytes in both matrices). Selectivity: no endogenous interference at the retention times of the analytes in plasma, whereas in blank urine salicyluric acid was observed in few humans as it is a normal constituent. Similarly in few urine blanks interference at the gentisic acid retention time as this interference is aglycone of endogenous glucuronide. In rabbit plasma there is no interference for aspirin and salicyluric acid, but showed salicylic acid (up to 0.2 mg/mL). Rabbit blank urine showed 625 and 619 mg/mL of salicylic acid and salicyluric acid, respectively, and interference During the extraction process the loss of aspirin was minimal (~2.2%) and the acidic pH condition of mobile phase also helped to reduce the aspirin hydrolysis. Authors reported that loss of salicylic acid owing to sublimation was also minimal, which was due to the components of plasma and urine. Application of this method was shown in a human PK study following oral administration of 975 mg aspirin tablet. The Cmax and Tmax observed for aspirin acid were 11.7 1.7 mg/mL and 20 min, respectively with a t of 30.6 min; for salicylic acid the Cmax and Tmax were found to be 56.7 4.8 mg/mL and 2.37 0.37 h, respectively with a t of 2.70 h. All the obtained values were in agreement with the earlier reported values. The recoveries of free salicyluric acid, conjugated salicyluric acid, free salicylic acid, conjugated salicylic acid and gentisic acid from human urine were 61.3, 7.4, 8.5, 9.6 and 1.1%, respectively. The method was also used to determine the PK of aspirin and its metabolites following i.v. R. Mullangi et al.
Analyte(s)
Authors
Mays et al., 1984
Table 1. (Continued) Sample processing details of gentisic acid was high in blank, which did not permit to quantitate gentisic acid. administration of aspirin at a dose of 25 mg/kg to rabbits. Chromatographic conditions Validation parameters Applicable conclusions
Analyte(s)
Authors
923
Mobile phase: isocratic mobile phase comprising 5 mM phosphate buffer (pH 2.5) MeOHACN, 68:16:16, v/v/v, at a ow-rate of 1.3 mL/min. Absolute recovery: from plasma the recovery for aspirin, salicylic acid and salicyluric acid was 92 1, 85 4 and 52 8%, respectively; whereas from urine the recovery for salicylic acid, salicyluric acid and gentisic acid was 98 4, 93 7 and 55 3%, respectively.
only 500 mL was analyzed in the similar lines, and the quantity of salt and phoshphoric acid reduce by half. Urine samples, in urine aspirin metabolites were analyzed by mixing 1 mL of urine with equal volume of 0.2 M acetate buffer (pH 5), a drop of chloroform and 20 mL of b-glucuronidase and incubated for 20 h at 37 C on a shaking water bath. Following incubation the solution was transferred into a 10 mL volumetric ask and the volume was made up with buffer and from this 1 mL was added into a centrifuge tube containing 12 mL of DCM (9 mg/mL) and the same amounts of salt and phoshphoric acid as above and processed in similar lines of plasma samples. For the estimation of salicylic acid and salicyluric acid the residue was reconstituted in 1 mL of mobile phase, whereas for estimation of entisic acid the reconsti-tution volume was 500 mL. Internal standard: mephenytoin. Detection: lmax set at 237 nm except for gentisic acid Stability: extracted samples in mobile phased showed 4%
(Continues)
924
Sample processing details estimation in urine for which the lmax set at 330 nm. Retention time: 4.5, 5.7, 7.4, 10.0 and 11.6 min for gentisic acid, salicyluric acid, aspirin, salicylic acid and IS, respectively. Volume of injection: 10 mL except for gentisic acid estimation in urine, where the injection volume was 50 mL. Total run time: 12 min. System: HPLC with UV detector. loss of aspirin at room temperature. Linearity: 0.110 and 0.140 mg/mL for aspirin and salicylic acid, respectively (r2 > 0.998 for both analytes). Absolute recovery: ~93 and 88% for aspirin and salicylic acid, respectively. Accuracy and precision: found to be within the acceptable limits. Chromatographic conditions Validation parameters Applicable conclusions Column: Hypersil ODS (250 4 mm, 5 mm) at ambient room temperature. Mobile phase: isocratic mobile phase comprising ACN5% acetic acid, 20:80 (v/v) at a ow-rate of 2.5 mL/min. Detection: lmax set at 237 nm. Retention time: 3.54 and 4.85 min for aspirin and salicylic acid, respectively. Volume of injection: 20 mL. Enzymatic hydrolysis of aspirin was reduced by processing the blood under chilled conditions and by addition of potassium uoride. Further the plasma samples were stable by storing them in deep freezer for 5 days. Even after storage for 4 weeks the degradation of aspirin was 510%. This method was used to quantitate both aspirin and salicylic acid in human plasma following oral administration of 500 mg of aspirin tablet. Matrix: human blood and plasma. Extraction: plasma was extracted with ether: hexane (80:20, v/v), vortex mixed and centrifuged. The organic layer was evaporated to dryness and reconstituted in mobile phase and used for HPLC analysis. Entire samples processing was done in an ice-bath in order to avoid the loss of salicylic acid by sublimation. Internal standard: no IS was used. Matrix: rat plasma and urine. System: HPLC with UV detector. As authors utilized direct precipitation, the variation in the data was considerably decreased. Extraction: plasma samples, an aliquot of plasma (50 mL) was precipitated with 100 mL of ACN containing IS, vortex mixed for 1 min and centrifuged for 5 Column: m Bondapak C18 maintained at ambient room temperature. R. Mullangi et al. Linearity: 0.5200 mg/mL (in plasma) and 6200 mg/mL (in urine) for all analytes in both matrices (r > 0.999 for all analytes in both matrices). Absolute recovery: the mean recovery was 61.6, 94.1, 99.5 and 90.7% for gentisic acid, salicyluric acid, aspirin and salicylic acid, respectively, in plasma; whereas the recovery of gentisic acid, salicyluric
Analyte(s)
Authors
Nieder and Jaeger, 1983
Aspirin, salicylic acid, salicyluric acid and gentisic acid
Bakar and Niazi, 1983
Table 1. (Continued) Sample processing details min at 15,000 rpm and the supernatant was used for HPLC analysis. Mobile phase: isocratic mobile phase comprising water MeOHglacial acetic acid, 64:35:1 (v/v/v) at a ow-rate of 2 mL/min. acid, O-anisic acid and salicylic acid was 97, 95, 92 and 91%, respectively, from urine. Accuracy and precision: found to be within the acceptable limits. Review of aspirin bioanalytical methods Chromatographic conditions Validation parameters Applicable conclusions
925
Analyte(s)
Authors
Urine samples, 1 mL of urine was acidied with 0.5 mL of 6 M HCl, then 6 mL of anhydrous ether was added and mixed for 15 min and centrifuged for 10 min at 2000 rpm and 5 mL of ether layer was separated. To the ether layer, 1 mL of 0.1 M (pH 7) phosphate buffer was added, mixed for 15 min and centrifuged for 10 min at 2000 rpm and the ether layer was aspirated and buffer solution was used for analysis. Internal standard: o-toluic acid (dissolved in waterACN, 2:1, v/v; 100 mg/mL IS solution for plasma linearity having concentration >20 and 10 mg/mL IS solution for plasma linearity having concentration <20 mg/mL) and oanisic acid (dissolved in water; 100 mg/mL IS solution for urine linearity having concentration >40 and 20 mg/mL IS solution for plasma linearity having Detection: lmax set at 238 and 305 nm for plasma and urine samples analysis, respectively. Retention time: 3.0, 3.7, 4.8, 5.0, 7.1 and 10.2 min for gentisic acid, salicyluric acid, aspirin, o-anisic acid, salicylic acid and o-toluic acid, respectively. Volume of injection: 20 mL. Total run time: ~12 min.
(Continues)
926
Sample processing details Chromatographic conditions Validation parameters Applicable conclusions concentration <40 mg/mL). Matrix: rabbit plasma, urine and tissue homogenate. Human plasma. System: HPLC with UV detector. Column: LiChrosorb RP18 (150 4 mm, 5 mm) maintained at 45 C. Linearity: 0.5250 mg/mL for aspirin and salicylic acid and 0.5100 mg/mL for salicyluric acid and gentisic acid (r > 0.993 for all analytes). Limit of detection: 0.5 and 2.5 mg/mL for aspirin and gentisic acid; 0.2 mg/mL for salicylic acid and salicyluric acid. Extraction: plasma samples, to an aliquot of plasma (200 mL), 50 mL concentrated phosphoric acid and 600 mL ethyl acetate were added, vortex mixed and centrifuged at 600 g for 10 min at 10 C. The supernatant (400 mL) was stored at 26 C until analysis. Before HPLC analysis the supernatant was dried in an ice-bath under a gentle stream of air and the residue was dissolved in 200 mL of mobile phase and 100 mL was used for HPLC analysis. Tissue homogenate, 500 mg of tissue was homogenized with 2 mL of distilled water and processed as described for plasma samples. Mobile phase: isocratic mobile phase comprising MeOH water, 40:60 (v/v), nal pH 3.0 (adjusted with 0.005 M phosphoric acid and sodium hydroxide) at a ow-rate of 1.5 mL/min. Enzymatic hydrolysis of aspirin was performed by collecting blood into tubes containing sodium uoride and heparin (4 mg of sodium uoride and 50 IU of heparin for 1.5 mL blood). The vials were kept on ice no longer than 30 min before processing. Plasma was harvested from blood by centrifuging at 1500 g for 10 min at room temperature. This method was used to quantitate aspirin, salicylic acid and salicyluric acid in rabbit plasma and tissues following i.v. administration of aspirin at 50 mg/kg dose. Gentisic acid could not be detected in plasma following aspirin administration. This method was also used to quantitate aspirin, salicylic acid and salicyluric acid in plasma samples collected from healthy human volunteers following oral administration of a 650 mg oral dose of aspirin effervescent tablet. Gentisic acid was not detected in human plasma. R. Mullangi et al. Urine samples, urine was 10-fold diluted with water and processed as Detection: lmax set at 280 nm. Absolute recovery: 93 4, 95 4, 102 3 and 101 3% for aspirin, salicylic acid, salicyluric acid and gentisic acid, respectively, from rabbit plasma. From human plasma the recovery was 98 3, 89 5, 94 3 and 101 2% for aspirin, salicylic acid, salicyluric acid and gentisic acid, respectively. Selectivity: no interference from ascorbic acid, codeine, caffeine, inulin,
Analyte(s)
Authors
Reidl, 1983
Table 1. (Continued) Sample processing details described for plasma samples. Chromatographic conditions Validation parameters Applicable conclusions
Analyte(s)
Authors
927
Internal standard: no IS was used.
Retention time: 2, 2.7, 3.7 and 5.5 min for gentisic acid, salicyluric acid, aspirin and salicylic acid, respectively. Volume of injection: 100 mL.
dextropropoxyphene and TEA at the retention time of the analytes. Paracetamol interfered with salicylic acid retention time. Stability: aspirin stability was assessed for 27 days at 5, 26 and 80 C and it was found that only at 80 C was little hydrolysis observed.
Aspirin, salicylic acid and salicyluric acid
Buskin et al., 1982
Matrix: human plasma and urine.
Extraction: plasma samples, to an aliquot of 1 mL plasma, 1 mL of aqueous oxalic acid (1 mol/L) and 1 mL of aqueous IS solution (0.7 mg) and 10 mL of etherhexane (1:1, v/v) were added vortex
Column: Spherisorb ODS (250 4.6 mm, 5 mm) maintained at ambient room temperature.
Linearity: 0.051 and 110 mg/L as low and high calibration curves for aspirin and salicyluric acid in plasma; 110 and 10100 mg/L as low and high calibration curves salicylic acid in plasma. Low and high calibration curves with concentration range of 10200 and 2002000 mg/L; 5100 and 100750 mg/L; 140 and 4040 mg/L for salicylic acid, salicyluric acid and gentisic acid, respectively. For total salicylates and gentisates the low and high calibration curve ranges were 20400 and 4004000 mg/L and 240 and 20400 mg/L, respectively. Precision and accuracy: found to be within acceptance limits.
Blood samples were collected into chilled tubes containing lithium heparin and potassium uoride (to prevent aspirin hydrolysis). Plasma samples were stored at 20 C until analysis. The method was applied to analysis of plasma samples collected from healthy human volunteers following oral administration of a 650 mg oral dose of aspirin.
(Continues)
928
Sample processing details Chromatographic conditions Validation parameters Applicable conclusions Mobile phase: isocratic mobile phase comprising water phosphate buffer (0.2 mol/L, pH 2.5)ACN, 35:40:25, v/v/v, nal pH 3.5) and water phosphate buffer (0.2 mol/L, pH 2.5)ACN, 35:50:20, v/v/v) for plasma and urine samples analysis, respectively, at a ow-rate of 1 mL/min. Stability: in the processed samples (maintained in autosampler and at room temperature) aspirin half-life was found to be 118 h. The concentration of aspirin decreased by 3 and 20% following storage at 20 C and analyzed on day 1/5 and day 11. mixed for 90 s and centrifuged at 500 g for 3 min. Following centrifugation 250 mL organic phase was taken out and to this 300 mL of 0.5 mol/L of phosphate buffer (KH2PO4: Na2HPO4, pH 7) was added, vortex mixed for 90 s and centrifuged for 3 min, then mix 200 mL aqueous phase with 200 mL 1 mol/L phosphate buffer (H3PO4KH2PO4, pH 2) and used for HPLC analysis. Urine samples, following spiking of IS solution (100 mg/L), extract one volume of urine with 4 volumes of ethyl acetate hexane (1:1, v/v). After back-extracting with 500 mL of the 0.5 mol/L phosphate buffer (pH 7), mix 200 mL of aqueous phase with 200 mL of 1 mol/L oxalic acid and use for HPLC analysis. To assay conjugated salicylate and gentisate in urine, 1 mL of urine was mixed with 1.5 mL of concentrated HCl in a loosely capped screwtop tube. Heat the contents to 120 C in an autoclave for 1 h. After cooling, 1 mL of 1 mol/L Detection: lmax set at 234 and 303 nm for plasma and urine samples analysis, respectively. R. Mullangi et al.
Analyte(s)
Authors
Table 1. (Continued) Sample processing details oxalic acid, 8 mL of ethyl acetatehexane (1:4, v/v) and IS solution [400 mg of IS in 200 mL of 25 mmol/L phosphate buffer (KH2PO4/ Na2HPO4, pH 7.0], vortex-mixed and centrifuged, then backextracted the organic phase with 300 mL of the 0.5 mol/L phosphate buffer (pH 7.0). Further processing and chromatography are as described above for urine samples but with only 360 mL of extract injected onto the HPLC column. Internal standard: m-hydroxybenzoic acid (for plasma samples) and 2-hydroxy-6-napthoic acid (for urine samples). Retention time: 5.6, 6.4, 8.9 and 10.6 min for IS, salicyluric acid, aspirin and salicylic acid, respectively, in plasma samples analysis but there was slight delay in elution of these compound during urine samples analysis. The IS used in urine samples analysis eluted at 22 min. The change in IS for urine samples was done due to endogenous interference at the retention time of IS used for plasma samples. Volume of injection: 20150 mL. Total run time: 12/25 min. System: HPLC with UV detector. Chromatographic conditions Validation parameters Applicable conclusions Review of aspirin bioanalytical methods
Analyte(s)
Authors
929
Aspirin, salicylic acid, salicyluric acid and gentisic acid Extraction: plasma samples, to an aliquot of plasma, 20 mL perchloric acid (30%) containing IS and
Rumble et al., 1981
Matrix: human plasma (200 mL) and urine.
Column: m Bondapak C18 (300 3.9 mm, 10 mm) coupled to a guard column (23 3.9 mm packed with m Bondapak
Linearity: 0.5200 mg/mL for all analytes in plasma (r > 0.99 for all analytes) and 2500 mg/mL for salicylic acid in urine. Limit of detection: 0.2, 0.5, 0.1 and 0.1 mg/mL for gentisic acid, salicyluric acid, aspirin and salicylic acid, respectively.
Blood samples were collected into chilled tubes containing anticoagulant and sodium uoride (to prevent aspirin hydrolysis). Application of this method was shown in a human PK study following oral administration of 1.2 g of soluble aspirin in 100 mL of (Continues)
930
Sample processing details C18/Porasil) maintained at ambient room temperature. Chromatographic conditions Validation parameters Applicable conclusions Mobile phase: isocratic mobile phase comprising ACN0.03% phosphoric acid (pH 2.5), 30:70 (v/v) at a ow-rate of 1 mL/min. 200 mL MeOH were added, vortex mixed for 2 min and centrifuged at 1500 g for 2 min and supernatant was used for HPLC analysis. Urine samples, total salicylate was estimated in urine resulting from the hydrolysis of salicylate conjugates. To an aliquot of urine (2 mL), 2 mL 10 M HCl was added in a glass ampoule and air was removed by ushing with oxygen before sealing. The sealed ampoule was autoclaved for 3 h at 120. On cooling, the contents were vortexed and 20 100 mL hydrolysate was made up to 300 mL with distilled water and to this 200 mL ACN was added, vortex mixed and centrifuged for 5 min at 1500 g. The supernatant was used for HPLC analysis. Internal standard: p-toluic acid (0.02%). Specicity: although there was an endogenous peak at 8.8 min, no interference at the retention times of the analytes in plasma and urine. Interference from other (~28 drugs) was studied by taking the plasma of patients taking various drug and found that they did not interfere with the assay. Detection: lmax set at 237 and 313 nm for plasma and urine samples analysis, respectively. Retention time: 5.2, 6.2, 8.0, 11.2 and 13.6 min for gentisic acid, salicyluric acid, aspirin, salicylic acid and IS, respectively. Volume of injection: 20 mL. Total run time: 15 min. System: HPLC with UV detector. water. Plasma concentrations of aspirin, salicylic acid, salicyluric acid and gentisic acid were measured. The method was also used to quantitate salicylic acid in urine. Although gentisic acid was detected in urine, it was poorly resolved from urine endogenous peaks and therefore not quantied. The aspirin quantied in plasma and salicylic acid in urine was comparable with earlier reported values following administration of a comparable dose of aspirin. R. Mullangi et al. Matrix: human serum (500 mL). Linearity: 0.062.4 and 0.052 m mol/L for aspirin and salicylic acid, respectively (r 2 > 0.999 for both analytes). Blood samples were collected from volunteers and patients. After centrifugation, plasma or serum was collected and stored at 20 C until analysis (not more than 7 days).
Analyte(s)
Authors
Wahlin-Boll et al., 1981
Table 1. (Continued) Sample processing details Extraction: an aliquot of serum was acidied with200 mL of 1 M H3PO4 and extracted with 2 mL of diethyl ether and the contents were centrifuged. Following centrifugation the clear supernatant was evaporated under gentle stream of nitrogen and the residue was dissolved in mobile phase. The clear supernatant was used for HPLC analysis. Internal standard: sodium paraaminosalicylate. Absolute recovery: 98.2106.9 and 92.5115.8% for aspirin and salicylic acid, respectively. Column: m Bondapak C18 (300 3.9 mm, 10 mm) maintained at ambient room temperature. Specicity: no endogenous interference at the retention times of the analytes in plasma. Authors also tested 50 drugs that can potential interfere the chromatography and found that none of them interfered with the assay. Chromatographic conditions Validation parameters Applicable conclusions Review of aspirin bioanalytical methods
Analyte(s)
Authors
931
Mobile phase: isocratic mobile phase comprising MeOH0.01 M KH2PO4 (pH 3.5), 40:60 (v/v) at a ow-rate of 2.6 mL/min. Detection: lmax set at 280 nm. Retention time: 4.75 and 7.0 min for aspirin, and salicylic acid, respectively. Volume of injection: 100 mL. Precision: found to be within acceptable limits.
Total run time: 20 min.
Cham et al., 1980
Matrix: human plasma.
Linearity: 2 30 and 5100 mg/L for aspirin and salicylic acid (r2 > 0.999 for both analytes).
Physostigmine (nal concentration 104 M) addition prevented the hydrolysis of aspirin in serum up to 7 days at 20 C, then progressive degradation was observed. Authors observed that addition of physostigmine to blood samples before centrifugation led to hemolysis, hence sodium uoride was used subsequently. This method was used to quantitate aspirin and salicylic acid from the serum/plasma collected from patients suffering with rheumatoid arthritis (received 4 0.75 g of aspirin) and healthy human volunteers (received 1 g of aspirin as a single dose). The serum collected from rheumatoid arthritis patients (following chronic administration of aspirin) showed salicylic acid (0.452.65 mmol/L) and no aspirin levels at steady state. The variability in salicylic acid levels at steady state was in accordance with earlier reports. Following oral administration of 0.5 g of aspirin to healthy human volunteers, aspirin was detectable until 35 h, whereas salicylic acid was seen up to 24 h. The mean peak concentrations of aspirin and salicylic acid in plasma were 0.09 and 0.45 mmol/L, respectively. This method was also suitable to quantitate serum concentrations of diusinal, indomethacin, indoprofen and indobufen under therapeutic conditions. Blood samples were collected into tubes containing heparin and physostigmine (to prevent aspirin (Continues)
932
Sample processing details Extraction: to an aliquot of plasma equal volume of methyl cyanide was added, vortex mixed and centrifuged at 900 g for 5 min and supernatant was used for HPLC analysis. Internal standard: no IS was used. Column: m Bondapak C18 (300 3.9 mm, 10 mm) maintained at ambient room temperature. Specicity: no endogenous interference at the retention times of the analytes in plasma. Authors also tested 50 drugs that can potential interfere the chromatography and found that none of them interfered with the assay. Absolute recovery: 98.2106.9 and 92.5115.8% for aspirin and salicylic acid, respectively. Chromatographic conditions Validation parameters Applicable conclusions hydrolysis) and spun within 15 min to harvest plasma. Plasma was snap frozen until analysis. Application of this method was shown in a human PK study following oral administration of 900 mg soluble and effervescent aspirin tablet. The Cmax and Tmax observed for aspirin acid were 11.7 32/.62 mg/L and 14.14 min, respectively with t of 13.1 min. The AUC01 was found to be 15.32 mg h/L. Mobile phase: isocratic mobile phase comprising acetic acidMeOHwater, 5:22:73, v/v/v) at a ow-rate of 2.6 mL/min. Detection: lmax set at 280 nm. Retention time: 4.75 and 7.0 min for aspirin, and salicylic acid, respectively. Volume of injection: 100 mL. Total run time: 20 min. System: HPLC with UV detector. Precision: found to be within acceptable limits. Linearity: 1150 mg/mL for salicylic acid and salicylsalicylic acid at lmax 300 nm (r2 > 0.994 for both analytes) and 2100 mg/mL for all analytes at lmax 280 nm. Matrix: human plasma and urine. Extraction: to an aliquot of plasma (500 mL) or urine (2 mL) IS solution, 0.9 mL of 0.27 M HCl and either 10 mL methylene chloride (for plasma samples) or hexane (10 mL) and the tubes were shaken for 15 min and centrifuged for 5 min at 750 g. The organic layer was evaporated to dryness and the residue was dissolved in 0.5 mL MeOH and used for analysis. Internal standard: a-phenylcinnamic acid Column: m Bondapak C18 (300 4 mm) maintained at ambient room temperature. R. Mullangi et al. Specicity: no endogenous interference at the retention A less polar solvent was used for extraction of urine to eliminate interfering substances. UV lmax set at 300 nm gave higher sensitivity for salicylsalicylic acid and salicylic acid but aspirin could not be monitored. For aspirin lmax 280 nm was ideal but at this lmax the sensitivity for salicylsalicylic acid and salicylic acid was 2- to 3-fold lower. This method was successfully used to quantitate salicylsalicylic acid and salicylic acid in human plasma and urine following oral administration of 1000 mg of salicylsalicylic acid (as two tablets) to healthy human volunteers. Cmax (33.4 mg/mL) of salicylsalicylic acid was attained at 1 h and the half-life was found to be 0.8 h. Similarly for the salicylic acid (which was released following in vivo hydrolysis of salicylsalicylic acid), the Cmax
Analyte(s)
Authors
Aspirin, salicylsalicylic acid and salicylic acid
Harrison et al., 1980
Table 1. (Continued) Sample processing details (100 mL of 0.1 mg/mL solution in MeOH). times of the analytes in both plasma and urine. Precision and accuracy: found to be within acceptable limits. (55.2 mg/mL) was attained at 4 h and the half-life was 3.5 h. The urinary excretion of salicylsalicylic acid and salicylic acid was 10 and 29.8 mg, respectively. Chromatographic conditions Validation parameters Applicable conclusions Review of aspirin bioanalytical methods
Analyte(s)
Authors
933
Aspirin and salicylic acid Column: Lichrosorb RP18 (250 4.6 mm, 10 mm) maintained at 40 C.
Lo and Bye, 1980
Mobile phase: isocratic mobile phase comprising MeOH1% acetic acid (60:40, v/v) at a ow-rate of 2 mL/min. Detection: lmax set at 300 nm for all analytes except for aspirin, which was detected at 280 nm. Retention time: 2.2, 3.0, 5.8 and 8.0 min for aspirin, salicylic acid, salicylsalicylic acid and IS, respectively. Volume of injection: 25 mL. Total run time: 9 min. System: HPLC with UV and uorescence detectors. Linearity: 120 and 2100 mg/mL for aspirin and salicylic acid (r2 > 0.9994 for all analytes). Specicity: no endogenous interference at the retention times of the analytes.
Extraction: to an aliquot of plasma, 300 mL of IS solution, 1 mL of 5% ortho-phosphoric acid (w/v, pH 1) and 10 mL chloroform were added, mixed and centrifuged for 10 min at 1000 g. The organic layer (9 mL) was evaporated under nitrogen in an ice water bath. The residue was dissolved in 200 mL of mobile phase and used for HPLC analysis. Internal standard: 3,4dimethylbenzoic acid (10 mg/mL in ACN). Mobile phase: isocratic mobile phase comprising MeOH0.072% orthophosphoric acid (55:45, v/v) at a ow-rate of 1.5 mL/min. Detection: lmax set at 234. Retention time: 2.75, 4.10 and 7.21 min for aspirin, salicylic acid and IS, respectively. Volume of injection: 10 mL. Total run time: 15 min.
Absolute recovery: >95 and 70% for aspirin and salicylic acid, respectively. Precision and reproducibility: found to be within acceptable limits.
Blood was collected in a plastic tube containing lithium heparin and potassium uoride (50 mL, 50% w/v) and kept in an ice-bath. Plasma was harvested by centrifuging at 2 C and stored at 20 C until analysis. This method was used to quantitate aspirin and salicylic acid in human plasma following oral administration of 600 mg (2 300 mg) of soluble aspirin tablets to healthy human volunteers. Aspirin and salicylic acid were quantiable up to 2 and 24 h, respectively, in plasma. The Cmax for aspirin and salicylic acid was found to be 14.4 and 46.8 mg/mL, respectively.. The t for aspirin was 15.5 min.
(Continues)
934
Sample processing details Matrix: human plasma and urine. System: HPLC with UV and uorescence detectors. Chromatographic conditions Validation parameters Applicable conclusions Column: m Bondapak C18 maintained at 50 C. Linearity: 0.55, 0.510 and 5 100 mg/mL for salicyluric acid, aspirin and salicylic acid in plasma, respectively (r2 > 0.9994 for all analytes). Similarly in urine: 0.220, 2100, 2200 and 204000 mg/mL for gentisic acid, aspirin, salicylic acid and salicyluric acid, respectively (r2 > 0.9996 for all analytes). Specicity: no endogenous interference at the retention times of the analytes in both plasma and urine except an interfering peak at retention time of gentisic acid in urine. Blood was collected in chilled (4 C) vacutaniers having 14 mg potassium oxalate and 17.5 mg sodium uoride, and plasma was harvested within 15 min. Similarly urine samples were acidied (75 mL of 7 M phosphoric acid to 4 mL urine). Both plasma and urine samples were stored at 30 C until analysis. This method was used to quantitate aspirin, salicylic acid, salicyluric acid and gentisic acid in human plasma and urine following oral administration of 650 mg of aspirin to healthy human volunteers. As a uorescence detector was used in tandem with a UV detector authors could quantify gentisic acid in plasma. Extraction: an aliquot of plasma (500 mL) was acidied with 200 mL of 7 M phosphoric acid to a pH of 1.5. To this IS solution was added and extracted with 6 mL of benzene: ethyl acetate (1:1, v/v) by vortexing for 1 min. The organic layer was separated following centrifugation for 5 min and evaporated under vacuum to avoid loss of salicylic acid by sublimation. The residue was dissolved in 2 mL of mobile phase and analyzed within 24 h. Urine samples were also analyzed in a similar way except 400 mL of urine was acidied with 75 mL of 7 M phosphoric acid. Internal standard: p-toluic acid (2 and 15 mg/mL for plasma and urine samples analysis, respectively). Mobile phase: isocratic mobile phase comprising 20% ACN in 1% glacial acetic acid in water at a ow-rate of 1.5 mL/min. Detection: lmax set at 280 nm and uorescence detector Precision and reproducibility: found to be within acceptable limits. R. Mullangi et al.
Analyte(s)
Authors
Amick and Mason, 1979
Table 1. (Continued) Review of aspirin bioanalytical methods Sample processing details Chromatographic conditions Validation parameters Applicable conclusions
935
Analyte(s)
Authors
Aspirin, salicylic acid and salicyluric acid Column: m Bondapak C18 maintained at 50 C.
Peng et al., 1978
Matrix: rabbit plasma.
excitation was set at 295 nm. Aspirin, salicylic acid (in plasma) and IS were detected under UV, whereas gentisic acid, salicylic acid (in urine) salicyluric acid were detected using uorescence detector. Retention time: 3.6, 4.7, 6.2, 7.7 and 12.4 min for gentisic acid, salicyluric acid, aspirin, salicylic acid and IS, respectively. Volume of injection: 300 and 100 mL for plasma and urine samples analysis, respectively. Total run time: 15 min. System: HPLC with UV detector. Linearity: 0.5300 mg/mL for all analytes (r2 > 0.999 for all analytes). Specicity: no endogenous interference at the retention times of the analytes in both rabbit and human plasma.
The method was successfully applied to an i.v. PK study in rabbits at a dose of 50 mg/kg of aspirin.
Extraction: to an aliquot of plasma (100 mL), IS solution was added and acidied with one drop of 85% H3PO4. This mixture was vortex mixed with 500 mL of benzene: ethyl acetate (1:1, v/v) for 30 s and centrifuged at 2000 rpm for 1 min. The organic layer was separated and dried under gentle steam of nitrogen using an ice batch (avoids sublimation of salicylic acid). The residue was reconstituted in mobile phase and used for HPLC analysis. Internal standard: phthalic acid. Mobile phase: isocratic mobile phase comprising 30% (v/v) ACN acetic acid in dilute
Recovery: were 8994% for the analytes. (Continues)
R. Mullangi et al. 2,3-DHBA, 2,3-Dihydroxybenzoic acid; ACN, acetonitrile; DCM, dichloromethane; F/T, freezethaw; HCl, hydrochloric acid; H3PO4, phosphoric acid; IS, internal standard; KH2PO4, potassium dihydrogen phosphate; MeOH, methanol; Na2HPO4, disodium hydrogen phosphate; PK, pharmacokinetic; SPE, solid-phase extraction; TEA, triethylamine; TFA, triuoroacetic acid. Liquidliquid extraction. A large number of solvents are available and there is sufcient literature-based data for nding suitable extraction conditions. LLE is exible, and offers opportunities for parallel processing of large numbers of samples. Regarding cost, LLE is less expensive than SPE. However, despite its widespread use, LLE is a tedious multistage operation, has problems with emulsion formation and invariably consumes large amounts of organic solvents. Off-line methodologies are often very tedious and time-consuming, and the risk of sample loss and/or cross contamination is high. Peng et al. (1978) reported a method for ASA, SA and SUA in plasma using a solution of benzene and ethyl acetate with UV detection at 237 nm. In 1979, Amick and Mason published a method for ASA, SA, SUA and GA from plasma and urine with the same extraction strategy (i.e. benzene and ethyl acetate) with UV detection at 280 nm for SA and ASA, and urometric detection for SUA and GA. The method had a lower limit of quantitation of 0.5 and 5 mg/mL for ASA and SA, respectively. Ethyl acetate extraction followed by UV detection at 280 nm was also used in method reported by Reidl (1983) for ASA, SA, SUA and GA from plasma, tissue and urine. Several methods have been reported for the use of chlorinated methane solvents as extraction solvent, e.g. dichloromethane (Harrison et al., 1980; Mays et al., 1984; Klimes et al., 1992) and chloroform (Lo and Bye, 1980). In addition to two methods discussed in beginning ethereal extraction approach was also followed by several groups (Wahlin-Boll et al., 1981; Nieder and Jaeger, 1983; Bakar and Niazi, 1983; Brandon et al., 1985; Siebert and Bochner, 1987; Shen et al., 1990; Bae et al., 2008). All of these methods were developed on a UV detector (229280 nm) for plasma and at 305 nm for urine (Bakar and Niazi, 1983), except for the reports of Siebert and Bochner (1987) and Bae et al. (2008). Siebert and Bochner (1987) used uorescence detection, whereas Bae et al. (2008) used mass spectrometric detection. While the majority of researchers followed a conventional evaporation strategy to concentrate the analytes (to obtain higher sensitivity), Buskin et al. (1982), Siebert and Bochner (1987) and Shen et al. (1990) used back-extraction using pH 7 buffer from organic media, thereby cleaning samples to reduce the baseline noise levels of analytes of interest. Also, Shen et al. (1990) approach prevented evaporation-related loss of ASA/SA as they underwent sublimation (Kwong, 1987; Gaspari and Locatelli, 1987; Shen et al., 1990; Klimes et al., 1992; Kees et al., 1996). More recently, Bae et al. (2008) published a method for the quantitation of both ASA and SA from human plasma utilizing etherethyl acetate (1:4, v/v) extraction (for ASA), and precipitation using acetonitrile (for SA). The range of quantitation for ASA was 5500 ng/mL and that for SA was 505000 ng/mL. The detection was performed on LC-MS/MS in negative mode and the assay was validated for accuracy, precision and stability studies. Solid-phase extraction. Solid-phase extraction is an increasingly useful sample preparation technique that has been commonly employed in bioanalytical work. With SPE, many of the problems associated with LLE can be prevented, such as incomplete phase separations, less than quantitative recoveries, the use of expensive and breakable specialty glassware and the disposal of large quantities of organic solvents. SPE is more efcient than LLE, yields quantitative extractions that are easy to perform, is rapid and can be easily automated to process large batches of samples. While it minimizes solvent use, it also improves the laboratory time utilization for scientists.
Applicable conclusions
Analyte(s)
Authors
Sample processing details
phosphoric acid (0.05%, pH 2.5 0.1) at a ow-rate of 1 mL/min. Detection: lmax set at 237 nm. Retention time: 3.7, 4.6 5.3 and 7 min for IS, salicyluric acid, aspirin and salicylic acid, respectively. Volume of injection: 50 mL. Total run time: 20 min.
Chromatographic conditions
Accuracy and precision: found to be within acceptable limits with CV <4% across the concentrations tested.
Validation parameters
936
Table 2. Summary of validation of various published LC-MS/MS methods Sample processing details Regression type: linear t with weighting factor of 1/x2 and 1/x for aspirin and salicylic acid, respectively. Calibration range: 3500 and 305000 ng/mL for aspirin and salicylic acid, respectively (r2 > 0.998 for both analytes). Matrix: 100 mL of rat plasma. Extraction: following addition of 20 mL of ACN the contents were vortexed and 300 mL of 0.1% formic acid ACN was added and centrifuged for 5 min, then 200 mL of supernatant was transferred into an analytical vial. Internal standard: 6methoxysalicylic acid (10 mL of 20 mg/mL). System: LC-ESI-MS/MS by MRM in negative mode. Column: Ultra C18 (100 2 mm, 3 mm) maintained at ambient room temperature. Mobile phase: isocratic mobile phase comprising ACNwater (63:37, v/v) containing 0.1% formic acid, delivered at a ow-rate of 0.3 mL/min. Injection volume: 5 mL. Mass spectrometry detection: aspirin, m/z 178.9 ! 136.8; salicylic acid, m/z 137 ! 93; and IS, m/z 167 ! 123. Retention time: 1.7, 2.2 and 2.4 for aspirin, salicylic acid and IS, respectively. Chromatographic conditions Validation parameters Applicable conclusions The validated method was applied to a rat pharmacokinetic (PK) study following oral and i.v. administration of aspirin at 1 mg/kg, and key PK parameters, viz. AUC0last, AUC01, Cmax, Tmax and t were calculated for aspirin and salicylic acid. Blood samples were collected into chilled tubes containing anticoagulant and 20 mL of 150 mg/mL potassium uoride (to minimize the hydrolysis of aspirin to salicylic acid) and centrifuged to harvest plasma. The plasma was stored at 80 C until analysis.
Analyte(s)
Authors
937
Xu et al., 2009
Total run time: 3 min.
Absolute recovery: mean recoveries were 61.1 4.47 64.4 4.96 and 27.631.8 for aspirin and salicylic acid, respectively. Specicity: no interference peaks were observed at the retention time of the analytes and IS from seven different sources of human plasma. Accuracy and precision: intraand inter-day accuracies for aspirin and acetyl salicylic acid were were 86.8106 and 98.0107%; 97.3105 and 98.8107%, respectively; the intra- and inter-day RSD for aspirin and acetyl salicylic acid were 2.1813.2 and 2.10 9.12%; 5.9712.5 and 3.05 8.12%, respectively. Stability studies: both aspirin and salicylic acid were found to be stable in the autosampler for 24 h; salicylic acid was found to be stable under F/T conditions and on the bench-top (24 h), whereas aspirin was found to be unstable under these two
(Continues)
938
Sample processing details Chromatographic conditions Validation parameters Applicable conclusions System: LC-ESI-MS/MS by MRM in negative mode. Column: Luna C18 (50 2 mm, 3 mm) maintained at ambient room temperature. Mobile phase: isocratic mobile phase comprising ACNwater (80:20, v/v) with 0.1% formic acid, delivered at a ow-rate of 0.2 mL/min. Injection volume: 5 mL. Mass spectrometry detection: aspirin, m/z 179 ! 137; salicylic acid, m/z 137 ! 93; and IS, m/z 435 ! 319. Retention time: 1.06, 1.0 and 1.67/1.72 for aspirin, salicylic acid and IS, respectively. Total run time: 3 min. conditions, hence the authors recommended processing aspirin samples under lower temperature and plasma samples should be thawed. Regression type: linear t with weighting factor of 1/x2 and 1/x for aspirin and salicylic acid, respectively. Matrix: 100 and 1000 mL of human plasma for salicylic acid and aspirin, respectively. Extraction: for aspirin, following addition of 20 mL of 50% potassium uoride (to minimize the hydrolysis of aspirin to salicylic acid) and IS solution and 1 mL of 50 mL formic acid (for acidication of plasma), the contents were vortex-mixed with 5 mL of a mixture of ethyl acetate and diethyl ether (4:1, v/v) and centrifuged for 10 min at 4 C. Following evaporation of organic solvent the residue was reconstituted with 100 mL of mobile phase. All the procedures were completed within 20 min at 4 C in an ice-bath. For salicylic acid, to the plasma, 400 mL of ACN containing IS solution was added and centrifuged for 10 min and the clear supernatant was used for analysis. Internal standard: simvastatin (10 mL of 20 mg/mL) Calibration range: 5500 and 505000 ng/mL for aspirin and salicylic acid, respectively (r2: 0.99 for both analytes). Absolute recovery: mean recovery was 61.1 4.47 64.4 4.96 and 27.631.8 for aspirin and salicylic acid, respectively. Specicity: no interference peaks were observed at the retention time of the analytes and IS from seven different sources of human plasma. Accuracy and precision: intraand inter-day accuracies for aspirin and salicylic acid were 86.589.4 and 95.0103%; 104108 and 91.298.5%, respectively; the intra- and inter-day RSD for aspirin and salicylic acid were were 1.50 4.30 and 1.507.90%; 6.40 9.30 and 6.07.80%, respectively. Stability studies: stock solutions of aspirin and salicylic acid were found to be stable for 7 days at 4 C; both aspirin and acetyl salicylic acid were found to be stable in human The validated method was successfully applied to a human PK study following administration of 100 mg enteric coated tablet of aspirin in Korean healthy volunteers and key PK parameters, viz. AUC0last, AUC01, Cmax, Tmax and t were calculated for aspirin and salicylic acid. Authors could quantify salicylic acid from the rst time point onwards, indicating that the conversion of aspirin to salicylic acid was rapid. The AUCsalicylic acid/AUCaspirin ratio was 61.3. R. Mullangi et al.
Analyte(s)
Authors
Bae et al., 2008
Review of aspirin bioanalytical methods ACN, acetonitrile; ESI, electro-spray ionization; IS, internal standard; F/T, freezethaw; IS, internal standard; MRM, multiple reaction monitoring; PK, pharmacokinetic; RSD, relative standard deviation. Several SPE methods have been reported for ASA and SA, but most of them lack complete validation data for the intended application. Pirola et al. (1998) published a method with C8 SPE material and the eluent was directly analyzed for ASA and SA in plasma at UV 234 nm. McMahon and Kelly (1998) and Abu-Qare and Abou-Donia (2001) published their approach for the same application utilizing C18 SPE cartridges, rst back-ushing retained analytes onto the analytical column, and other methods involved evaporation of the methanolic eluent to concentrate the analytes prior to injection on the analytical column. However, the two methods did not include the addition of IS to the sample matrix. All of the above methods had a lower limit of quantitation of 100 ng/mL for ASA or higher. Yamamoto et al. (2007) published an on-line MC-SAX SPE method for ASA and SA in plasma with UV detection at 235 nm. The use of this method provided a much higher sensitivity for both analytes (i.e. the lower limit of quantitation attained for ASA and SA was 60 ng/mL). Internal standard selection Determination of the drug concentration in biological matrix, either in drug discovery or drug development stages, is usually a considerable challenge in terms of both sample preparation and chromatographic optimization (Srinivas, 2006). With recent advancements in mass spectrometry, enabling higher sensitivity to be attained, suitable IS is of the utmost importance. From the published literature, it was evident that several methods did not include an IS (Cham et al., 1980; Reidl, 1983; Siebert and Bochner, 1987; Krivoskov et al., 1996; McMahon and Kelly, 1998; AbuQare and Abou-Donia, 2001), and all of these methods utilized UV detection. Considering the polar nature of analytes, i.e. ASA (pKa 3.5) and other metabolites, most researchers preferred to use acidic substances, e.g. p-toluic acid (Amick and Mason 1979; Rumble et al., 1981; Bakar and Niazi 1983; Gaspari and Locatelli, 1987), benzoic acid and its analogs (Lo and Bye 1980; Buskin et al., 1982; Nieder and Jaeger 1983; Kees et al., 1996), as candidates for IS. Other examples include the use of simvastatin (pKa 5.5; Bae et al., 2008) and a structure analog of SA (Xu et al., 2009) on a mass spectrometer. The selection of acid substrate as an IS was thought to provide the right balance across the conditions of processing, chromatography and detection employed for the measurements of ASA, SA and other metabolites. Chromatography In simplistic terms, it involves passing a mixture dissolved in a mobile phase through a stationary phase, which separates the analyte to be measured from other molecules in the mixture based on the differential partitioning effect between the mobile and stationary phases. Subtle differences in a compounds partition coefcient result in differential retention on the stationary phase, thus accounting for separation of the two peaks. Reverse-phase chromatography has been widely used, in which the liquid mobile phase is water (aqueous component) combined with an organic solvent such as methanol or acetonitrile and the stationary phase surface is nonpolar or hydrocarbon-like. The choice of reverse-phase systems was obvious since ASA, being a small molecule (i.e. small carbon chain, C9H8O4) with a low log P (1.39) and a polar surface area of 63.6 2 would evidently lead to poor retention on conventional reverse-phase columns. To retain the analytes of interest to a greater extent, most researchers
Applicable conclusions Table 2. (Continued) Analyte(s) Authors Sample processing details Chromatographic conditions plasma after short-term storage (30 min for in an icebath for aspirin and 24 h at room temperature for salicylic acid), for three F/T cycles and for 12 h at 4 C in autosampler. Validation parameters
939
R. Mullangi et al. have used octadecyl silane as the stationary phase, i.e. traditional reverse phase matrix. Because it has the highest degree of hydrophobicity, C18 silica interacts with the widest range of compounds and the interactions are generally more pronounced. Some authors have used C8 (OKruk et al., 1984; Gaspari and Locatelli, 1987; McMahon and Kelly, 1998). McMahon and Kelly (1998) reported their observation of the superior retention of ASA and SA on C18 phase vs other tested materials. Shen et al. (1990) used a phenyl column with ion-pairing agent triethylamine as an additive in mobile phase methanol and water in proportions of 280:717.5:2.5 (v/v/v, pH 3.5). associated metabolites). Hence, it is not only important but highly recommended to ensure that the validity of using the published assay for ASA and/or its metabolites is veried when a newer agent that has not been used before is combined with the ASA therapy. Multiple reaction monitoring mode on mass spectrometers provides a selective signal of the analyte of interest with enhanced sensitivity, and this be useful for ASA and SA analysis if the matrix is loaded with other co-administered agents that may confound the chromatography and/or other modes of detection. The same procedure may also serve in the interest of the co-administered drugs.
Discussion
Given the popularity of ASA and its metabolites, the bioanalytical arena in the last few decades has witnessed continuous publications regarding the quantitation of ASA and its metabolites in biological matrices. Owing to inherent problems associated with bioanalysis of ASA, SA and other metabolites of SA, viz. lack of stability, low molecular weight and high polarity, it is of prime importance that a proper strategy and framework be developed before initiation of such assays. It is important to note that, irrespective of the chosen matrix, one has to be cautious of ex vivo degradation either enzymatically or nonenzymatically of ASA and its metabolites. As discussed, the hydrolysis of ASA to SA by esterase enzyme can be arrested by addition of a nonspecic esterase inhibitor, e.g. potassium uoride, and it can be further reduced if necessary by lowering both pH and temperature. While for sample cleanup precipitation can be thought the easiest and cheapest option, it should be emphasized that care should be taken in selecting the appropriate precipitating medium to avoid any matrix effects. On the other hand, LLE clearly provides a larger option of different individual solvents and solvent mixtures of varying degrees of polarity, but the LLE option may produce inconsistent recoveries and can be tedious and difcult to automate. SPE as compared with other extraction techniques can produce substantially cleaner extracts with avoidance of any signicant matrix effect, and ease of automation is also enhanced. Invariably, the detection system employed depends on the level of sensitivity and selectivity needed for the selectivity. In this regard, the availability of recent LC-MS/MS methods for ASA and its metabolites facilitate accurate monitoring and/or quantication for the effective management of the ongoing ASA therapy. As ASA is typically administered in a poly-pharmacy situation with patients having diabetes, high lipids, metabolic syndrome and cardiovascular risk factors, one has to be cognizant that the developed LC-MS/MS must have sufcient selectivity and specicity to precisely monitor the levels of ASA and/or its metabolites in the presence of other co-administered drugs and associated metabolites [ACE (angiotensin converting enzyme) inhibitors (captopril, enalapril, lisinopril, rampipril), acetaminophen, atenolol, butalbital, caffeine, clopidogrel, dipyridamole, ephedrine, esomeprazole, heparin, losartan, metoclopramide, paracetamol, phenacetin, prasugrel, prostacyclin, statins (atorvastatin, pravastatin, simvastatin), telmisartan, ticlopidine, vitamin K antagonists (acenocoumarol, dicoumarol, warfarin), etc]. By the same token, one has to ensure that the methods developed for monitoring other co-administered drugs do not inuence ASA. Because ASA and its metabolites show complexity in their physicochemical properties, this may heighten issues related to extraction and/or chromatography when analyzed with other agents (and/or
References
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Review of HPLC Methods & HPLC-MS Detection For Direct Determination of Aspirin With Its Metabolites in Biological Matrices

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Special issue: review

(wileyonlinelibrary.com) DOI 10.1002/bmc.2694

Biomed. Chromatogr. 2012; 26: 906941

Copyright 2012 John Wiley & Sons, Ltd.

Review of aspirin bioanalytical methods

Aspirin or acetyl salicylic acid (ASA)

Salicylic acid (SA)

Salicyluric acid (SUA)

conjugation with glucuronic acid

Acyl and phenyl glucuronides of salicylic acid

conjugation with glycine

Gentisic acid (GA)

conjugation with glucuronic acid

Gentisic acid glucuronide

Gentisuric acid (GUA)

Figure 1. Metabolic pathway of aspirin.

Biomed. Chromatogr. 2012; 26: 906941

Copyright 2012 John Wiley & Sons, Ltd.

Table 1. Summary validation of various published HPLC methods

Aspirin, salicylic acid, 2,3DHBA and gentisic acid

Yamamoto et al., 2007

Copyright 2012 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 2012; 26: 906941

Biomed. Chromatogr. 2012; 26: 906941

Review of aspirin bioanalytical methods

Abu-Qare and Abou-Donia, 2001

Copyright 2012 John Wiley & Sons, Ltd.

Aspirin and salicylic acid

McMahon and Kelly, 1998

Copyright 2012 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 2012; 26: 906941

Biomed. Chromatogr. 2012; 26: 906941

Review of aspirin bioanalytical methods

Aspirin and salicylic acid

Pirola et al., 1998

Matrix: human skin and plasma.

System: HPLC with UV detector.

Copyright 2012 John Wiley & Sons, Ltd.

Copyright 2012 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 2012; 26: 906941

Aspirin and salicylic acid

Kees et al., 1996

Biomed. Chromatogr. 2012; 26: 906941

Copyright 2012 John Wiley & Sons, Ltd.

Volume of injection: 10 mL.

Aspirin, salicylic acid and salicyluric acid

Krivoskov et al., 1996

Copyright 2012 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 2012; 26: 906941

Aspirin, salicylic acid and gentisic acid

Klimes et al., 1992

Biomed. Chromatogr. 2012; 26: 906941

Review of aspirin bioanalytical methods

Copyright 2012 John Wiley & Sons, Ltd.

Copyright 2012 John Wiley & Sons, Ltd.

Aspirin, salicylic acid, gentisic acid and salicyluric acid

Shen et al., 1990

Biomed. Chromatogr. 2012; 26: 906941

Biomed. Chromatogr. 2012; 26: 906941

Review of aspirin bioanalytical methods

Aspirin and salicylic acid

Siebert and Bochner, 1987

Matrix: human plasma (1 mL).