1 of 179 Complete Record Title: High-throughput quantification for a drug mixture in rat plasma -a comparison of Ultra Performance.(TM).

liquid chromatography/tandem mass spectrometry with high-performance liquid chromatography/tandem mass spectrometry. Author(s): Yu, K.; Little, D.; Plumb, R.; Smith, B. Journal: Rapid Communications in Mass Spectrometry Volume: 20 Issue: 4 Page(s): 544-552 Publication Date: 30 Jan 2006 Abstract: A quantitative Ultra Performance.(TM). liquid chromatography/tandem mass spectrometry (UPL/MS/MS) protocol was developed for a five-compound mixture in rat plasma. A similar high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) quantification protocol was developed for comparison purposes. Among the five test compounds, three preferred positive electrospray ionization (ESI) and two preferred negative ESI. As a result, both UPLC/MS/MS and HPLC/MS/MS analyses were performed by having the mass spectrometer collecting ESI multiple reaction monitoring (MRM) data in both positive and negative ion modes during a single injection. Peak widths for most standards were 4.8 s for the HPLC analysis and 2.4 s for the UPLC analysis. There were 17 to 20 data points obtained for each of the LC peaks. Compared with the HPLC/MS/MS method, the UPLC/MS/MS method offered 3-fold decrease in retention time, up to 10fold increase in detected peak height, with 2-fold decrease in peak width. Limits of quantification (LOQs) for both HPLC and UPLC methods were evaluated. For UPLC/MS/MS analysis, a linear range up to four orders of magnitude was obtained with r2 values ranging from 0.991 to 0.998. The LOQs for the five analytes ranged from 0.08 to 9.85 ng/ml. Three levels of quality control (QC) samples were analyzed. For the UPLC/MS/MS protocol, the percent relative standard deviation (RSD%) for low QC (2 ng/ml) ranged from 3.42 to 8.67% (N = 18). The carryover of the UPLC/MS/MS protocol was negligible and the robustness of the UPLC/MS/MS system was evaluated with up to 963 QC injections.

2of 179 Complete Record Title: Determination of benzodiazepines in human urine using solid-phase extraction and high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. Author(s): Hegstad, S.; Oiestad, E. L.; Johansen, U.; Christophersen, A. S. Journal: Journal of Analytical Toxicology Volume: 30 Issue: 1 Page(s): 31-37 Publication Date: Jan 2006 Abstract:

A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed and validated for the determination of benzodiazepines, on the market in Norway, and/or their metabolites in human urine. The following compounds were included: 7-aminonitrazepam, 7-aminoclonazepam, 7aminoflunitrazepam, alprazolam, alpha.-hydroxyalprazolam, oxazepam, 3-OH-diazepam, and ndesmethyldiazepam. The method includes hydrolysis of urine samples (0.5 mL) with .beta.-glucuronidase at 60 .degree.C for 2 h before solid-phase extraction with a polymer-based mixture mode column. The analytes were quantified in multiple reaction monitoring mode using two transitions. Deuterated analogues were used as internal standards for all analytes except 7-aminonitrazepam and .alpha.-hydroxyalprazolam, which were quantified using 7-aminoclonazepam-d4 and alprazolam-d5, respectively. The concentration range was 0.1-8.0 .mu.M for 7-aminonitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, alprazolam and .alpha.hydroxyalprazolam and 0.5-40 .mu.M for the other compounds. The average recovery for the different analytes ranged from 56% to 83%. The between-day precision of the method was in the range of 3-12%. The limits of quantification were found to be between 0.002 and 0.01 .mu.M for the different compounds. Comparison with other analytical methods was performed for method validation, using approximately 500 samples provided by the routine laboratory at the Norwegian Institute of Public Health. The LC-MS-MS method has proven to be robust and specific for the determination of benzodiazepines in urine. It has been routinely used for approximately 1800 samples in the past 7 months. 3 of 179 Complete Record 68-24-G-10093 Title: Screening analysis of benzodiazepine medicines and selected metabolites in plasma and urine using gas chromatography with nitrogen phosphorus detection Author(s): Jiang, Z. L.*; Tan, J. Y.; Yao, L. J.; Xing, L. M. Journal: Fenxi Kexue Xuebao Volume: 21 Issue: 6 Page(s): 639-642 Publication Date: Dec 2005 Abstract: A screening analytical method for 11 benzodiazepine medicines and their main metabolites in plasma and urine using gas chromatography with nitrogen phosphorus detection was developed in this paper. The plasma was extracted at pH 10.8 with benzene-isoamlyalcohol (98.5 : 1.5). The urine was hydrolysed with .beta.gluculonidase and then extracted as plasma. Gas chromatography was performed on two columns of different polarity. The limits of detection of most benzodiazepines in specimens were better than 10 ng/mL. This method was suitable for quantitative analysis of benzodiazepine and was used to measure the benzodiazepine concentrations of specimens collected from subjects digested therapeutic dose medicines. The results indicated that this method can be easily used in drug examination for the case of drug-facilitated robbery. 9 of 179 Complete Record 68-16-G-10126 Title: Screening method for benzodiazepines and hypnotics in hair at pg/mg level by liquid chromatographymass spectrometry/mass spectrometry.

Author(s): Villain, M.; Concheiro, M.; Cirimele, V.; Kintz, P. Journal: Journal of Chromatography, B: Analytical Technologies in the Biomedical and Life Sciences Volume: 825 Issue: 1 Page(s): 72-78 Publication Date: 15 Oct 2005 Abstract: A procedure is presented for the screening of 16 benzodiazepines and hypnotics in human hair by LCMS/MS (alprazolam, 7-aminoclonazepam, 7-aminoflunitrazepam, bromazepam, clobazam, diazepam, lorazepam, lormetazepam, midazolam, nordiazepam, oxazepam, temazepam, tetrazepam, triazolam, zaleplon and zolpridem). The method involves decontamination of hair with methylene chloride, hair cut into small pieces, incubation of 20 mg in phosphate buffer (pH 8.4) in the presence of 1 ng diazepam-d5 used as internal standard, liquid-liquid extraction with diethyl ether/methylene chloride (10/90) and separation using liquid chromatography-tandem mass spectrometry. The limits of quantification for all benzodiazepines and hypnotics range from 0.5 to 5 pg/mg using a 20 mg hair sample. Linearity is observed from the limit of quantification of each compound to 200 pg/mg (r2 > 0.99). Coefficients of variation measured on six points and at two concentrations (10 and 50 pg/mg) range from 5 to 20% for all drugs but one. Extraction recovery, measured at the two same concentrations range from 32 to 76%. These results were found suitable to screen for 16 benzodiazepines in hair and detect them at very low concentrations, making this method suitable to monitor single dose.

11 of 179 Complete Record 68-12-G-10225 Title: Identification of alprazolam in hair in two cases of drug-facilitated incidents. Author(s): Kintz, P.*; Villain, M.; Cheze, M.; Pepin, G. Journal: Forensic Science International Volume: 153 Issue: 2-3 Page(s): 222-226 Publication Date: 29 Oct 2005 CODEN: Abstract: The use of a drug to modify a person's behaviour for criminal gain is not a recent phenomenon. However, the recent increase in reports of drug-facilitated crimes (sexual assault, robbery) has caused alarm by the general public. Among the drugs that can be used, alprazolam (Xanax), an anxiolytic benzodiazepine, has been seldom observed. To document two cases involving this drug, we have developed an approach based on hair testing by LC-MS/MS. After pH 8.4 buffer incubation and extraction with methylene chloride/diethyl ether (80/20, v/v), hair extracts were separated on a XTerra MS C18 column using a gradient of acetonitrile and formate buffer. Alprazolam and diazepam-d5, used as internal standard, were detected by electrospray tandem mass spectrometry.

13 of 179 Complete Record Title: Determination of four benzodiazepines residues in pork using gas chromatography-mass spectrometry. Author(s): Wang, L. P.; Li, X.; Sun, Y.; Zhao, H. X.; Qiu, Y. M.*; Zhong, W. K.; Tang, Y. Z.; Wang, D. N.; Zhou, Z. Q. Journal: Fenxi Huaxue Volume: 33 Issue: 7 Page(s): 951-954 Publication Date: Jul 2005 Abstract: Solid phase extraction (SPE) and gas chromatography-mass spectrometric method have been developed for the determination of diazepam, estazolam, alprazolam and triazolam residues in pork. The conditions for extraction and cleanup on C18 column of benzodiazepines were investigated and optimized. The analytes were separated by HP-5 chromatographic column, detected by mass detector in electron impact and time program-selected ion monitoring mode (EI/SIM) and quantified with the external standard calibration curve method. Linear calibration curves were obtained in the concentration ranges from 5 to 100 .mu.g/L for diazepam and from 50 to 1000 .mu.g/L for estazolam, alprazolam and triazolam (the correlation coefficients were above 0.99). The average recoveries for the four benzodiazepines spiked in pork ranged from 60% to 115% and their relative standard deviations were between 3.8% and 19.7%. The limits of detection were 2 .mu.g/kg for diazepam and 10 .mu.g/kg for estazolam, alprazolam and triazolam in pork. 18 of 179 Complete Record Title: Screening and confirmatory method for benzodiazepines and hypnotics in oral fluid by LC-MS/MS. Author(s): Kintz, P.; Villain, M.; Concheiro, M.; Cirimele, V. Journal: Forensic Science International Volume: 150 Issue: 2-3 Page(s): 213-220 Abstract: A procedure is presented for the screening of 17 benzodiazepines and hypnotics in oral fluid after collection with the Intercept.RTM. device by LC-MS/MS (alprazolam, 7-aminoclonazepam, 7-aminoflunitrazepam, bromazepam, clobazam, diazepam, lorazepam, lormetazepam, midazolam, nordiazepam, oxazepam, temazepam, tetrazepam, triazolam, zaleplon, zopiclone and zolpidem). The method involves extraction of 0.5 mL of oral fluid (previously stored in the Intercept blue buffer) treated with 0.5 mL of phosphate buffer (pH 8.4) in the presence of 5 ng diazepam-d5 used as internal standard, with 3 mL of diethyl ether/methylene chloride (50/50) and separation using liquid chromatography-tandem mass spectrometry. The limits of quantification for all benzodiazepines and hypnotics range from 0.1 to 0.2 ng/mL. Linearity is observed from the limit of quantification of each compound to 20 ng/mL (r2 > 0.99). Coefficients of variation at 2 ng/mL, measured on 6 points range from 4 to 8% for all drugs, except zopiclone (34%). Extraction recovery, measured at the same concentration was higher than 90%. Ion suppression was evaluated for each compound and was

lower than 10% for all drugs except zopiclone (93%). These results were found suitable to screen for 17 benzodiazepines in oral fluid and detect them at very low concentrations, making this method suitable for monitoring subjects under the influence. 20 of 179 Complete Record Title: Simultaneous quantification of alprazolam, 4-and .alpha.-hydroxyalprazolam in plasma samples using liquid chromatography mass spectrometry. Author(s): Allqvist, A.; Wennerholm, A.; Svensson, J.-O.; Mirghani, R. A.* Journal: Journal of Chromatography, B: Analytical Technologies in the Biomedical and Life Sciences Volume: 814 Issue: 1 Page(s): 127-131 Publication Date: 5 Jan 2005 Abstract: A sensitive and specific method was developed for quantification of alprazolam and its two metabolites 4hydroxyalprazolam and .alpha.-hydroxyalprazolam in plasma. The work up procedure was solid phase extraction. Liquid chromatography-mass spectrometry (LC-MS) was used for separation, detection and quantification of the analytes. The limit of quantitation (LOQ) was 0.05 ng/ml for alprazolam and the two metabolites. The extraction recovery was more than 82% for alprazolam and its metabolites. The within-and between-assay coefficients of variation were in the range of 1.9-17.9%. The method was used for determination of the pharmacokinetics parameters of alprazolam and its two metabolites in healthy Caucasian subjects who ingested 1 mg of alprazolam. 23 of 179 Complete Record Title: NP -HPLC monitoring of benzodiazepine in human plasma. Author(s): Mao, G. F. Journal: Yaowu Fenxi Zazhi Volume: 24 Issue: 4 Page(s): 385-387 Publication Date: 31 Jul 2004 Abstract: To establish an NP -HPLC method for monitoring concentration of midazolam, estazolam, triazolam and alprazolam in human plasma. The plasma samples were extracted with ether. The organic phase was evaporated under nitrogen stream at 55 .degree.C in water bath. The remains were dissolved by absolute ethanol. Column: Shim -pack CLC -CN, 150 mm x 6.0 mm, 5 .mu.m; mobile phase: n-hexane -absolute ethanol -methanol (76 : 18 : 6) at a flow rate of 1.10 ml/min. The column temperature was 40.degree.C, UV detection wavelength was 230 nm. The contents of four components could be determined simultaneously. The linear ranges of midazolam, estazolam, triazolam and alprazolam were 0.050-20.0 .mu.g/ml(r >= 0.9996). The accuracies were 98.9%, 99.8%, 101.8%, 100.5% (n= 6). Intra-day RSD and inter-day RSD were less than 5.1%

(n= 6). The method is sensitive, specific, accurate and convenient for monitoring concentration of midazolam, estazolam, triazolam and alprazolam in human plasma. 28 of 179 Complete Record Title: Determination of the benzodiazepine plasma concentrations in suicidal patients using a radioreceptor assay. Author(s): de Jong, L. A. A.; Verwey, B.; Essink, G.; Muntendam, A.; Zitman, F. G.; Ensing, K.* Journal: Journal of Analytical Toxicology Volume: 28 Issue: 7 Page(s): 587-592 Publication Date: Oct 2004 Abstract: Impairments in memory and psychomotor function appear to be induced by benzodiazepines not only after long-term use, but also after administration of a single dose. Because it is known on which neurotransmitter system the benzodiazepines exert their action, the use of a quantitative radioreceptor assay (RRA) can be a useful tool in studying the inter-relationship between the neurochemical and memory process. The RRA measures the sum of the main compound(s) and all active metabolites present, where it relates the biological activity to the pharmacodynamic effect instead of relating it to the plasma levels of the individual compounds. In order to correlate the loss of memory with th benzodiazepine concentration, plasma concentrations were determined in suicidal patients. From suicidal patients (n = 84), the benzodiazepines in plasma were measured with a direct RRA using tritiated flunitrazepam as the labelled ligand. The receptor material was a lyophilized preparation from calf cortex. Furthermore, the samples were subjected to HPLC analysis, and the HPLC data were converted to diazepam equivalents using the cross-reactivities of the individual compounds. Patients who had ethanol residues in their plasma were excluded from this correlation experiment. The data (n = 40) obtained with the two analytical techniques were compared and correlated to assess the validity of the RRA in establishing the relationship between the loss of memory and the total amount of benzodiazepines present. The cumulative amount of diazepam determined with the RRA and the sum of compounds determined with the HPLC method, after correction using the cross-reactivities, were plotted and correlated using regression analysis. Regression analysis showed a x variable of 0.75 and a correlation coefficient of 0.67. The intercept was not significantly different from zero (P = 0.49, t-test), whereas the slope was significantly different from zero (P < 0.01). Benzodiazepines can be directly determined in plasma using the described RRA. The data obtained from HPLC analysis were easily converted to diazepam equivalents using the crossreactivities. A discrepancy between the data obtained from the two analytical techniques, however, indicates that certain metabolites are present, which were not quantified in the HPLC analysis, but were measured in the RRA. Therefore, the RRA proved to be a valuable tool for the assessment of clinical effects such as the demonstration of the loss of memory in suicidal patients after a benzodiazepine overdose. 35 of 179 Complete Record Title: Rapid analysis of benzodiazepines in whole blood by high-performance liquid chromatography: use of a monolithic column.

Author(s): Bugey, A.; Staub, C.* Journal: Journal of Pharmaceutical and Biomedical Analysis Volume: 35 Issue: 3 Page(s): 555-562 Publication Date: 28 May 2004 Abstract: In a previous work [J. Pharm. Biomed. Anal., 2000, 23, 447]a rapid HPLC method, using a monolithic column coupled with a diode-array detector, was developed for the quantitative determination of benzodiazepines in whole blood. The present method has been applied to the assay of eight benzodiazepines amongst the most frequently encountered in forensic toxicology: clonazepam, desalkylfurazepam, diazepam, flunitrazepam, lorazepam, midazolam, nordiazepam and oxazepam. The sample pretreatment involved a liquid-liquid extraction of blood samples by n-butyl chloride. The separation was carried out in reversed-phase conditions using a Chromolithtrade; Performance (RP-18e 100 x 4.6 mm) column. The mobile phase was composed of a phosphate buffer (35 mM, pH 2.1) and acetonitrile (7:3, v/v) and the flow-rate was 2 ml/min. The duration of the analysis was less than 4 min and the results of validation, including linearity, precision, recovery, limit of quantification, were satisfactory. The therapeutic and toxic concentrations usually encountered for these substances could be measured. The compounds were separated by a monolithic column which, on account of its particular structure, could bear higher flow-rates than usually found for this kind of analysis. The present method has been applied to two real cases and was tested with about 30 compounds. 40 of 179 Complete Record Title: Development and validation of a new HPLC analytical method for the determination of alprazolam in tablets. Author(s): Perez-Lozano, P.; Garcia-Montoya, E.; Orriols, A.; Minarro, M.; Tico, J. R.; Sune-Negre, J. M. Journal: Journal of Pharmaceutical and Biomedical Analysis Volume: 34 Issue: 5 Page(s): 979-987 Publication Date: 10 Mar 2004 Abstract: A new analytical method is developed together with its latter validation study, by means of a high resolution liquid chromatography (HPLC) of reverse phase to quantify alprazolam (8-chloro-1-methyl-6-phenyl-4H[1,2,4]triazole [4,3-.alpha.]-[1,4]benzodiazepine) in tablets. Determination is carried out by means of an ODS C18 column (200 mm x 4.6 mm i.d., 5 .mu.m particle size); the mobile phase consisted of a mixture of 0.02 M buffer solution of phosphates (pH 6.0) and acetonitrile (45:55, v/v). It is pumped through the chromatographic system at a flow rate of 0.50 ml/min. The UV detector is operated at 254 nm. The validation study is carried out fulfilling the ICH guidelines in order to prove that the new analytical method, meets the reliability characteristics, and these characteristics show the capacity of an analytical method to keep, throughout the time, the fundamental criteria for validation: selectivity, linearity, precision, accuracy and

sensitivity. The method is applied during the working day for the quality control of commercial alprazolam tablets in order to quantify the drug and its degradation products and to check the content uniformity test.

41 of 179 Complete Record Title: Detection of the adulteration of traditional alcoholic beverages by the separation and determination of alprazolam, chloral hydrate and diazepam using reversed-phase high-performance liquid chromatography. Author(s): Rao, R. N.; Parimala, P.; Khalid, S.; Alvi, S. N. Journal: Analytical Sciences Volume: 20 Issue: 2 Page(s): 383-386 Publication Date: Feb 2004 Abstract: A simple, rapid and reliable reversed-phase high-performance liquid chromatographic method for the separation and determination of psychotropic substances viz., alprazolam, chloral hydrate and diazepam in traditional alcoholic beverages, such as toddy, has been developed. Separation was accomplished using a reversed-phase Cis column with water-methanol-acetic acid (35:65:0.1 v/v/v) as a mobile solvent and a photodiode array detector at 210 nm. The limits of detection of alprazolam, chloral hydrate and diazepam were determined to be 0.8, 4.5 and 0.4 .mu.g, respectively. The validity of the method was checked by analyzing nearly 200 samples collected from different outlets by enforcement authorities, and the extent of adulteration was determined. 54 of 179 Complete Record Title: Spectrofluorimetric assay for the photodegradation products of alprazolam. Author(s): Nudelman, N. S.; Gallardo Cabrera, C. Journal: Journal of Pharmaceutical and Biomedical Analysis Volume: 30 Issue: 3 Page(s): 887-893 Publication Date: 15 Oct 2002 Abstract: A new spectrofluorimetric assay for the photodegradation products of the anxiolytic drug alprazolam is described. Alprazolam was found to be highly photolabile and special care should be taken to avoid light exposure during alprazolam storage and handling. The photostability of alprazolam was evaluated at pH 2.0, 3.6 and 5.0. The drug was exposed to UVA-UVB radiations, the photodegradation of alprazolam was followed by HPLC and the developed spectrofluorimetric assay allowed determination of the photodegradation products at very low concentrations (>= 10-5M). The photoinstability was found to increase with the pH value decreasing, consequently acidic media should be avoided during the drug-development process.

61 of 179 Complete Record Title: Identification of estazolam, alprazolam and triazolam in human urine by LC-MSn. Author(s): Gu, J. K.; Xia, R.; Zhong, D. F.; Sun, L. Journal: Yaoxue Xuebao Volume: 37 Issue: 2 Page(s): 138-140 Publication Date: Feb 2002 Abstract: Urine sample from subjects administered 4 mg dose of estazolam (I), alprazolam (II) and triazolam (III) were purified by SPE on on a Sep-Pak C18 column and eluted with 2 ml methanol. A 20 .mu.l portion of the solution was analysed by HPLC-MS on a 5 .mu.m Kromasil C18 steel stainless column (15 cm x 4.6 mm i.d.) with methanol H2O (4:1) as mobile phase (0.4 ml/min). The structures of I-III were identified by comparison of the observed mass spectra and the chromatographic retention time with those of the reference substance. The MS analysis was performed in positive mode and in two scan modes including SIM and full scan MS-MS mode. 91 of 179 Complete Record Title: Studies on determining the degree of dissolution of alprazolam tablets. Author(s): Wang, X. Q.; Wang, Q. R.; Wen, J. W. Journal: Yaowu Fenxi Zazhi Volume: 20 Issue: 1 Page(s): 53-56 Publication Date: 31 Jan 2000 Abstract: Two tablets were agitated in 100 ml HCl (9:1000) at 75 rpm for 30 min and filtered. The solution was diluted by a suitable amount and the absorbance was measured at 264 nm. The concentration of alprazolam (I) was calculated (equation given). The calibration graph for I was linear up to 26 mg/l. The solution was stable for 24 h. The recoveries were 100.5-107.8% with RSD of 1.6-2.7%. There was little interference from excipients. Results were compared with those obtained by HPLC. 93 of 179 Complete Record Title: Quantitation of alprazolam and .alpha.-hydroxyalprazolam in human plasma using liquid chromatography electrospray ionization MS-MS. Author(s): Crouch, D. J.; Rollins, D. E.; Canfield, D. V.; Andrenyak, D. M.; Schulties, J. E. Journal: Journal of Analytical Toxicology Volume: 23 Issue: 6 Page(s): 479-485 Publication Date: Oct 1999

Abstract: A sensitive and specific electrospray ionization high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) method has been developed for the quantitative determination of alprazolam (AL) and .alpha.-hydroxyalprazolam (OH-AL) in plasma. After the addition of deuterium labeled internal standards of AL and OH-AL, plasma samples were buffered to alkaline pH and extracted with toluene/ methylene chloride (7:3). Dried extract residues were reconstituted in HPLC mobile phase and injected onto a reversed-phase C18 HPLC column. The analytes were eluted isocratically at a flow rate of 250 .mu.L/min using a solvent composed of methanol and water (60:40) containing 0.1% formic acid. The analyses were performed using selected reaction monitoring. The assay was sensitive to 0.05 ng/mL. The intra-assay RSD were all <=5.6% for AL at 2, 5, and 20 ng/ml. At these concentrations, and all OH-AL intra-assay RSD were <=8.4%. The inter-assay RSD for AL were 11.8, 8.7 and 8.7%, at 2.0, 5.0, and 20.0 ng/mL, respectively. The OH-AL inter-assay RSD were 9.6, 9.2 and 7.8%, respectively, at the same concentrations. The assay accuracy was less than or equal to +/- 6.6% for both analytes at the three concentrations. The method was used to quantitate AL and OH-AL in plasma samples collected from 10 subjects who were administered a 1-mg oral dose of AL. The mean AL concentration peaked at 11.5 ng/ml 1 h after the dose and AL was detectable for 48 H. The mean OH-AL concentration peaked at 0.18 ng/mL 4 h after the dose and was undetectable by 36 h. Hydrolysis of the plasma samples had little effect on the detected AL concentrations but increased OH-AL concentrations substantially. Plasma/blood ratios for AL and OH-AL exceeded 1 in the study samples. 110 of 179 Complete Record Title: Determination of alprazolam and .alpha.-hydroxyalprazolam in plasma by high- performance liquid chromatography. Author(s): Nagasaki, T.; Ohkubo, T.; Sugawara, K.; Yasui, N.; Otani, K.; Kaneko, S. Journal: Analytical Sciences Volume: 13 Issue: 2 Page(s): 245-249 Publication Date: Apr 1997 Abstract: Plasma (1 ml) was mixed with internal standard (estazolam, 10 ng/10 .mu.l in methanol) and diluted to 5 ml with 1M-NaOH. The mixture was applied to a Sep-Pak CN cartridge activated with 5 ml acetonitrile and H2O. After washing with 10 ml H2O, the analytes were eluted with 5 ml aqueous 20% acetonitrile. The eluate was evaporated to dryness at 60.degree.C and the residue was dissolved in 50 .mu.l methanol and 100 .mu.l mobile phase. A 100 .mu.l portion of the resulting solution was analysed by HPLC on a 5 .mu.m Develosil C85 column (15 cm x 4.6 mm i.d.) with a mobile phase of 0.5% KH2PO4 of pH 4.5/acetonitrile (7:3) as mobile phase of pH 4.5 (1 ml/min) and detection at 230 nm. Calibration graphs were linear from 1-50 and 0.5-10 ng/ml of alprazolam (I) and its active metabolite, .alpha.-hydroxyalprazolam (II), respectively. Recoveries were 98.05+/-5.75% and 96.25+/- 5.7% of I and II, respectively, with corresponding RSD of <7.8 and 10.6%. The method was suitable for drug monitoring of I and II in plasma.

111 of 179 Complete Record Title: A fatality due to alprazolam intoxication. Author(s): Jankins, A. J.; Levine, B.; Locke, J. L.; Smialek, J. E. Journal: Journal of Analytical Toxicology Volume: 21 Issue: 3 Page(s): 218-220 Publication Date: May-Jun 1997 Abstract: Blood (2 ml) was mixed with 50 .mu.l chlordiazepoxide in methanol (100 mg/l) and 2 ml sodium borate. Tissue (1 g) was vortexed in 4 ml H2O. Homogenates were applied to a Chem Elut SPE column. Alprazolam (I) and .alpha.-hydroxyalprazolam (II) were eluted with 2 x 7 ml CH2Cl2. The eluent was evaporated to dryness and the residue was redissolved in 400 .mu.l methanol. A portion was injected into a 5 .mu.m Beckman ODS column (15 cm x 4.6 mm i.d.) with methanol/H2O (3:2) with 1% diethylamine of pH 6 as mobile phase (1.5 ml/min) and detection at 240 nm. The tissue distribution of I and II was tabulated for a fatal case due solely to the ingestion of alprazolam. Drug II was detected only in subclavian blood, urine, bile, and liver. 112 of 179 Complete Record Title: Detection of alprazolam in hair by negative-ion chemical-ionization mass spectrometry. Author(s): Hoeld, K. M.; Crouch, D. J.; Wilkins, D. G.; Rollins, D. E.; Maes, R. A. Journal: Forensic Science International Volume: 84 Issue: 1-3 Page(s): 201-209 Publication Date: 17 Jan 1997 Abstract: Chopped hair (10-25 mg) was treated with 5 ng of [2H4]triazolam (internal standard) and heated with 2 ml of 1M-NaOH at 40.degree.C overnight. The solution was adjusted to pH 9 with HCl, treated with 1 ml of saturated borax buffer solution and extracted with 7 ml of toluene/CH2Cl2 (7:3). The extract was evaporated in a current of air and the residue was heated with 25 .mu.l of ethyl acetate and 25 .mu.l of bis(trimethylsilyl)trifluoroacetamide (containing 1% of chlorotrimethylsilane) at 80.degree.C for 30 min. Portions (1 .mu.l) were analysed on a Restek-200 column (15 m x 0.25 mm i.d.; film thickness 0.25 .mu.m) with H2 as carrier gas (no flow rate given), temperature programming from 190.degree.C (held for 1 min) to 320.degree.C (held for 2 min) at 20.degree.C/min and negative-ion CIMS detection with CH4 as reagent gas. Ion currents were measured at m/z 308 and 310 for alprazolam (I) and internal standard, respectively. Calibration graphs were linear for 0.025-0.25 ppm of I in hair. The recoveries of 0.025 and 0.15 ppm of added I averaged 99.8% and 92.2%, respectively. The intra-and inter-assay RSD at these levels were 11.1-11.2% and 5.3-5.4%, respectively (n = 5). The method was applied to a study of the incorporation of I into the hair of rats fed with the drug.

118 of 179 Complete Record Title: X-ray investigations on three crystalline forms of alprazolam. Author(s): Reck, G.; Thiel, W.; Bahr, F.; Sauer, W.; Scharfenberg-Pfeiffer, D.; Landgraf, K.-F. Journal: Pharmazie Volume: 51 Issue: 8 Page(s): 553-558 Publication Date: Aug 1996 Abstract: An Enraf-Nonius CAD-4 diffractometer was used to examine the .alpha.- and .beta.- forms and dihydrate of alprazolam (I) and the results were processed by computer. The atomic co-ordinates and isotropic temperature factors of the atoms other than H are reported, as are the bond lengths and bond angles. The arrangements of the molecules in the crystals are shown. The calculated and experimental powder diffractogram data for .alpha.-I and the calculated data for the other forms are tabulated. Problems in unambiguously identifying the various forms are discussed. The .beta.-form was shown to be the most stable at room temperature; the .alpha.-form, although a metastable high-temperature form, showed no transformation into the .beta.-form after storage for 1 year. 123 of 179 Complete Record Title: High-performance liquid chromatography of alprazolam in postmortem blood using solid-phase extraction. Author(s): Hall, M. A.; Robinson, C. A.; Brissie, R. M. Journal: J. Anal. Toxicol. Volume: 19 Issue: 6 Page(s): 511-513 Publication Date: Oct 1995 Abstract: Whole blood (2 ml), 20% liver homogenate in H2O or 1% gastric contents in H2O was vortex mixed with 4 ml acetone for 30 s, allowed to stand for 10 s and then vortex mixed for 30 s. The mixture was centrifuged and 4 ml was evaporated to dryness under He at 70.degree.C. The residue was reconstituted in 1 ml 0.1Mphosphate buffer of pH 6.4, sonicated and vortex mixed. SPE and HPLC analysis was performed as described by Bio-Rad (Hercules, CA, USA; cf. Mazhar and Binder, J. Chromatogr., 1987, 797, 201) using the company s reagents, column and internal standard. The calibration graph was linear from 20-200 ng/ml and the limit of detection was 18 ng/ ml. RSD were 4-9.7% (n = 5). Recoveries were 73-94%. 131 of 179 Complete Record Title: Determination of alprazolam and its major metabolites in serum micro-samples by high-performance liquid chromatography and its application to pharmacokinetics in rats.

Author(s): Jin, L.-Y.; Lau, C. E. Journal: J. Chromatogr. B, Biomed. Appl. Volume: 654 Issue: 1 Page(s): 77-83 Publication Date: 18 Mar 1994 Abstract: To determine alprazolam (I) and its metabolites, rat serum (50 .mu.l) was mixed with 25 .mu.l of demoxepam soln. (0.5 .mu.g/ml; internal standard), 100 .mu.l of 1M-borate buffer of pH 9 and 2 ml of ethyl ether, centrifuged and the organic phase was evaporated to dryness under N2. The residue was redissolved in 50 .mu.l of methanol/acetonitrile/43mM-sodium acetate of pH 2.4 (45:8:47; buffer A) and portions (20 .mu.l) were analysed by HPLC on a column (15 cm .times. 2 mm i.d.) of Ultrasphere C18, with a Rheodyne 2 .mu.m pre-column filter, buffer A as mobile phase (0.3 ml/min) and detection at 230 nm. Triazolam (II) and its metabolites were determined using a mobile phase containing 40% methanol. The calibration graph was linear from 0.05-1 .mu.g/ml of I, II and their metabolites and the detection limit for each compound was 0.1 ng. The within- and between-day RSD were 1.92-6.87% and 2.43-9.04%, respectively, for I and its metabolites and 1.7-5.51% and 1.93-6.94%, respectively, for II and its metabolites; the recoveries were >82%. The method was applied to pharmacokinetic studies of I in rats. 133 of 179 Complete Record Title: Investigation on the thin-layer chromatography of product-related impurities in alprazolam. Author(s): Wang, Z. H.; Cui, Z. Q. Journal: Yaowu Fenxi Zazhi Volume: 14 Issue: 1 Page(s): 51-52 Publication Date: Jan 1994 Abstract: Alprazolam was dissolved in CHCl3 to a concentration of 40 mg/ml and then 10 .mu.l of the soln. was spotted on a silica gel GF254 plate alongside a standard soln. for TLC with CHCl3/methanol (9:1) as mobile phase. The chromatogram was examined under UV light at 254 nm. Two impurity spots were observed which accounted for 1%. All spots were well separated with no tailing. 139 of 179 Complete Record Title: Thin-layer chromatography scanning in measuring alprazolam concentration in plasma. Author(s): Zhang, A. H.; Bu, Z. Y.; Zhang, Y.; Wang, Q.; Li, W. B.; Qi, L. Journal: Zhongguo Yaoxue Zazhi Volume: 28 Issue: 4 Page(s): 233-234 Publication Date: April 1993 Abstract:

Heparin-treated blood (1 ml) was mixed with 3 ml of CHCl3 and then centrifuged at 3000 rpm for 5 min; the CHCl3 extract was evaporated (at 70.degree.) and the residue was taken up in 100 .mu.l of CHCl3. A portion (10 .mu.l) of the soln. was spotted on to a silica gel GF 254 plate for TLC with cyclohexane - CHCl3 diethylamine (50:40:11) as mobile phase. Alprazolam on the chromatogram was determined by using a Shimadzu CS-930 thin-layer scanner with measurement at 246 nm (reference at 320 nm). The calibration graph was rectilinear from 0.05 to 4 .mu.g of alprazolam and the coeff. of variation was 4.1%; recovery averaged 96.2%.