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Clinical Biochemistry 37 (2004) 175 – 183 Urinary screening for methylphenidate (Ritalin) abuse: a comparison
Clinical Biochemistry 37 (2004) 175 – 183 Urinary screening for methylphenidate (Ritalin) abuse: a comparison

Clinical Biochemistry 37 (2004) 175 – 183

Clinical Biochemistry 37 (2004) 175 – 183 Urinary screening for methylphenidate (Ritalin) abuse: a comparison of

Urinary screening for methylphenidate (Ritalin) abuse: a comparison of liquid chromatography–tandem mass spectrometry, gas chromatography–mass spectrometry, and immunoassay methods

J. Eichhorst, a M. Etter, a J. Lepage, a and D.C. Lehotay a,b, *

a Saskatchewan Health Provincial Laboratory, Regina, SK, Canada b Department of Pathology, University of Saskatchewan, Saskatoon, SK, Canada

Received 11 July 2003; received in revised form 4 November 2003; accepted 4 November 2003


Objective: To develop a routine method for detecting methylphenidate (Ritalin) use among drug abusers using liquid chromatography – tandem mass spectrometry (LC/MS/MS). The new methodology was designed to replace less reliable and/or more expensive and time- consuming techniques (GC/MS and ELISA) currently employed in our laboratory, and to provide a combined one-step screening and confirmation LC/MS/MS method. Design and methods: Because methylphenidate abuse is very prevalent in Saskatchewan, there is a demand to provide high volume urine screening both to detect abuse, and to monitor compliance. Random urine samples sent for drugs of abuse testing, standards, and controls were diluted 1:100 in methanol. Diluted specimens were injected directly into an Agilent 1100 liquid chromatograph coupled to a Sciex API 2000 mass spectrometer. The method utilized selected reaction monitoring (SRM) as well as an electrospray ionization source (EIS) to detect both urinary methylphenidate and the more prevalent metabolite, ritalinic acid (RA). Results: There appeared to be little or no sacrifice in sensitivity because the higher dilutions exhibited much less matrix effect. Limit of quantitation (LOQ) for methylphenidate was 100 nM and 500 nM for RA. Linear calibration curves from 100 to 1000 nM for Ritalin and 500 to 5000 nM for RA were acquired. Imprecision of spiked and true specimens did not exceed 10% and at the LOQ, it was less than 20%. Conclusions: A rapid, sensitive, reliable, and highly specific method by LC/MS/MS for detecting methylphenidate and its metabolite, RA, were developed. Both the cost and performance of the LC/MS/MS method were superior to GC/MS or ELISA, and it allows use of a single rapid procedure for both screening and confirmation. D 2003 The Canadian Society of Clinical Chemists. All rights reserved.

Keywords: Urinary screening; Methylphenidate; Ritalin


Methylphenidate (MPH) (Ritalin) is a phenethylamine derivative used in the treatment of depression, narcolepsy, attention-deficit disorder, and childhood hyperkinesis [1] . It is a central nervous stimulant that acts by inhibition of the presynaptic uptake of dopamine and norepinephrine and by promoting the synaptic release of dopamine [2] . Recently, MPH has become a popular drug of abuse with the usual mode of administration being intravenous injection of dissolved tablets often in combination with the drug pen-

* Corresponding author. Saskatchewan Health Provincial Laboratory, 3211 Albert Street, Regina, SK, Canada S4S 5W6. E-mail address: (D.C. Lehotay).

tazocine. Pentazocine (Talwin, Talacen) is a synthetic ben- zomorphan derivative that has properties of an analgesic and is about one third to one sixth as potent as morphine. The combination of Ritalin and Talwin is commonly referred to on the street as ‘‘poor man’s heroin’’ or ‘‘T’s and R’s’’. The fact that MPH is commonly prescribed in the treatment of attention-deficit disorder provides a likely source for drug abusers. Prescribing physicians have legit- imate concerns about whether or not the drug is being administered to children or instead being sold or used improperly by parents and guardians. In the past few years, our Toxicology department has become burdened with many requests for MPH analysis both for establishing abuse and monitoring compliance. By far the largest demand for

0009-9120/$ - see front matter D 2003 The Canadian Society of Clinical Chemists. All rights reserved.



J. Eichhorst et al. / Clinical Biochemistry 37 (2004) 175–183

this testing is from drug treatment centres, where the population is predominantly adult drug abusers. There seems to be a localized preference for MPH as a drug of abuse in Saskatchewan. Most drug treatment centres con- sider it to be as popular in our province as marijuana and/or opiate abuse. The volume of requests has steadily increased over the past 2 years to approximately 250 per week. This requires high volume capabilities. MPH is rapidly biotransformed in the body by hydrolysis of the methyl ester linkage to ritalinic acid (RA), an inactive metabolite. This hydrolysis also occurs in aqueous alkaline solutions and during incubation with blood or plasma [1] . Because about 70% (60 –81%) of MPH is eliminated in the urine as RA, it is obviously a better indicator (more prevalent) than the parent MPH for detecting usage. Ideally, any assay used for urinary MPH screening should be able to detect both parent and major metabolite. The R,R V isomer of MPH reportedly hydrolyses more rapidly than the S,S V isomer [3] ; however, we do not attempt to distinguish between these compounds. For some applications, it may be necessary to differentiate between both enantiomers of MPH as previously reported [4] , but for our purposes, this was not required. Precautions were taken to minimize spontaneous production of RA in specimens and standards by refrigeration and freezing if applicable. Previous methods employed by our laboratory included gas chromatography (GC), gas chromatography –mass spec- trometry (GC/MS), and an enzyme immunoassay (ELISA). The oldest method using GC was only used sparingly and will not be discussed here as a significant procedure. Access to instrumentation such as liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) has made possible the development of high-throughput analyt- ical methods that also provide excellent sensitivity and specificity. Trace level determination of drugs and/or metab- olites in biological matrices is an ongoing challenge in today’s clinical toxicology laboratory. Traditional techni- ques such as the above-mentioned GC/MS and ELISA are very useful, but in the case of MPH detection in urine specimens, both have drawbacks. The method developed for GC/MS provided adequate sensitivity and specificity (using selected ion recording—SIR); however, it required in- volved, labour intensive sample preparation, and with 15 – 20 min run times, was very slow. The ELISA method was found to be problematic in that it produced an unacceptably high number of false-positive results. Applying the urine specimens directly to the 96-well plates provided a false- positive (when compared to GC/MS) rate of approximately 10%. A modified method, which involved a simple solvent extraction, diminished the false-positive rate to approxi- mately 5%, which was somewhat of an improvement. The obvious advantage over the GC/MS methodology was speed and throughput in spite of poor selectivity. Selected reaction monitoring (SRM) LC/MS/MS is a reliable technique with high selectivity and sensitivity. Analysis for analytes in bio-matrices using LC/MS/MS

has become the method of choice for many laboratories where such instrumentation is available. A method was developed which utilized SRM of both MPH and RA in urine specimens with almost no sample preparation. The urine samples, standards, and controls are simply diluted in methanol containing an appropriate internal standard. The dilution is done directly in a 96-well plate and mixed for 15 s before placement on an Agilent 96-well plate auto- sampler. This is part of an Agilent 1100 HPLC system coupled directly to an API 2000 tandem mass spectrome- ter. Fifteen microliters of diluted specimen is injected directly through the autosampler system into the electro- spray source of the mass spectrometer without any chro- matographic separation. Run times are 1.6 min per sample, which allows an entire 96-well plate to be processed in approximately 4 h. Sample preparation for 80 samples takes only 30 min and runs can be processed overnight if instrument scheduling is a problem.


Methods and materials

Pure standards were obtained directly from pharmaceu- tical companies—MPH (Novartis), RA (Novartis), and mepivacaine (Abbott) (Fig. 1) . Levallorphan was utilized as an internal standard for the GC/MS procedure and was obtained from Roche. HPLC grade methanol, methylene chloride, and isopro- panol were obtained from Fisher Scientific and HPLC grade acetonitrile (Accusolve) and ethyl acetate were obtained

acetonitrile (Accusolve) and ethyl acetate were obtained Fig. 1. Structures of MPH, RA, and internal standard

Fig. 1. Structures of MPH, RA, and internal standard mepivacaine.

J. Eichhorst et al. / Clinical Biochemistry 37 (2004) 175–183


from Anachemia Canada Inc. Ammonium hydroxide was purchased from J.T. Baker, USA. The distilled water was purchased locally from Arctic Glacier Inc. and the 96-well flat-bottomed plates were purchased from Sarstedt Inc., USA. The plates were sealed with common aluminium foil. All pipetting was done using adjustable Gilson pipettes (Mandel Scientific). Certified drug-free urine was purchased from Bio-Rad Laboratories, Canada. All patient urine speci- mens were acquired from random urine drug screening samples submitted to the Saskatchewan Provincial Labora- tory. Solid-phase extraction columns (World Wide Monitor- ing Clean Screen ZSDAU020) were purchased from Diagnostix Inc., Canada. Both MSTFA ( N-methyl- n -trime- thylsilyl-trifluoroacetamide) and MBTFA [ N-methyl-bis(tri- fluoroacetamide)] were purchased from Pierce, USA and supplied by Chromatographic Specialties, Canada. Di-sodi- um hydrogen orthophosphate (dibasic) and sodium dihy- drogen orthophosphate (monobasic) buffers were purchased from BDH Chemicals, Toronto. Single-step ELISA Plate kits for MPH (catalogue # 600515) were purchased from Diagnostix Ltd., Mississauga, Canada.

QA material

All standards and controls were prepared using Bio-Rad drug-free urine. Separate stock solutions (4.0 mM) of MPH and RA were prepared in methanol and water, respectively. Urine standards were spiked at four levels of MPH and RA and run highest to lowest concentration both at the begin- ning and end of the run. Controls were prepared at or near the proposed cut-off level for each analyte (100 nM MPH and 1000 nM RA) as well as at a higher level more likely to be encountered in real screening situations. These were run every 20 samples or about 5 times on a 96-well plate. Standards were followed by blank samples and preceded by double blank samples. For LC/MS/MS analysis, standard curves for MPH included 1000, 500, 200, 100 nM standards and a blank. Standard curves for RA included 8000, 4000, 2000, 1000, 500 nM and a blank. Mepivacaine was used as an internal standard at a concentration of 0.03 A M in methanol. Both positive and negative urine samples were spiked at two levels to assess recovery. The spikes were at or near the limit of quantitation (LOQ) for each compound and at a higher level more realistic of urine samples from drug abusers. For GC/MS analysis, 4000, 2000, 1000 nM standards and blank samples spiked in drug-free urine were run. QC samples were spiked at 1000 nM. Confirmations of positive samples were based on the presence of RA because of its prevalence. A stock solution of 200 A M levallorphan was used as an internal standard. For the ELISA assay, a positive and negative control was run which the manufacturer provided. We also spiked drug- free urine at 100 and 1000 nM MPH to run as controls. The theoretical cut-off was established at 100 nM.

Sample preparation

LC/MS/MS Two microliters of urine was carefully pipetted into the bottom of the wells on a standard 96-well plate. Standards, blanks, controls, and spiked samples were treated similarly. To each well, 200 A l of internal standard (0.03 AM mepi- vacaine in methanol) was added. The plate was placed on a Wallac Delfia plate shaker for 20 s, covered with aluminium foil, and placed on the Agilent 1100 autosampler.

GC/MS An extraction and modified two-step derivatization was performed similar to previously reported methodologies [5] (Fig. 2) . Five milliliters of urine was spiked with 100 A l of internal standard (80 A M levallorphan) and buffered with 1 ml 0.1 M phosphate buffer (pH = 7.0). Tubes were vortexed and centrifuged at 2000 rpm for 5 min. Samples, standards, and controls were extracted using ‘‘Clean Screen’’ drugs of abuse cartridges. The extraction consisted of a column conditioning step using methanol, water, and phosphate buffer (pH 7.0). The samples were loaded, the column washed with water, 100 mM HCl and finally with methanol. After drying, the analytes were eluted with methylene chloride/isopropanol/ammonium hydroxide (78:20:2). Elu- ent was blown to dryness with nitrogen at <40 j C. To the dry residue, 80 Al MSTFA was added, the samples vortexed and heated at 60 j C for 5 min. The tubes were cooled to room temperature and 30 Al MBTFA was added. After vortexing and heating at 60 j C for 20 min, the samples were analyzed by GC/MS.

ELISA To remove interfering substances that caused a significant number of false-positive results, a simple extraction proce- dure was performed. To 0.5 ml samples, standards, and controls, we added 0.1 ml NH 4 OH and 2 ml chlorobutane. The tubes were vortexed vigorously for 15 s and centrifuged at 2000 rpm for 5 min. The solvent layer was removed and blown to dryness using nitrogen at <40 j C. To the dried

removed and blown to dryness using nitrogen at <40 j C. To the dried Fig. 2.

Fig. 2. Structures of MPH and RA derivatives.


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extract, 0.6 ml phosphate buffer was added and the tubes were vortexed vigorously for 5 s. The ELISA protocol was followed from this point on [6]. One hundred microliters of phosphate buffer was added to each well of a 96-well plate. Twenty microliters of sample, standards, or controls were pipetted into the appropriate wells. One hundred microliters of enzyme conjugate solution was added to each well and the plate incubated for 30 min at room temperature. The plate was then washed 6 times and 150 A l of substrate added to each well. After a 30-min incubation at room temperature, the plate was read on a microplate reader at a wavelength of 650 nm.

5 min. Data acquisition and analysis was done using Finnigan MassLab software v. 1.4.

ELISA The ELISA analysis was performed using 96-well plate format kits for MPH. All pipetting was done manually with

single and multichannel pipettes and the plates were read on

a Labsystems Multiscan RC well plate spectrophotometer

(Wallac, Canada). Multicalc software was used to control the spectrophotometer.


Results and discussion


Fig. 3 shows an acquired set of SRM scans from a ‘‘real’’

samples were sufficiently clean to be analyzed directly in the

The MS/MS analysis was performed using an Agilent

positive urine specimen. It is well known that reduced


LC system including a vacuum degasser, a binary

electrospray sensitivity is normally caused by the presence

pump, a well-plate auto-sampler, and a heated column

of endogenous components in biological matrices such as

compartment. This was coupled to an AB MDS Sciex API

urine. Such components will indeed cause ion suppression


triple quadrupole mass spectrometer. No chromato-

especially if insufficient separation or no chromatography is

graphic separation was performed. The mobile phase was 80% acetonitrile and 20% water containing 0.01% formic acid. The mobile phase flow was 250 A l/min and the injection volume was 15 Al. A 10-s flush-port needle rinse was incorporated into the procedure to prevent cross-con- tamination. SRM analysis was performed using the Sciex TurboIonSpray source at a temperature of 250 j C in positive ion mode. The mass spectrometer parameters were optimized using ‘‘Quantitative Optimization’’ in the Analyst 1.2 Soft- ware. The source parameters were optimized by infusing a solution of 1.0 AM MPH, RA, and internal standard in mobile phase at a rate of 10 Al/min using a Harvard syringe pump. The source parameter settings were as follows: curtain gas = 25, source temperature = 250 j C, source gas 1 = 40 psi, source gas 2 = 40 psi, ion spray voltage = 5300 V, CAD gas = 4, CE = 30 eV. Q1 and Q3 were operated at unit mass resolution. The SRM mass transitions monitored were 220.3 > 84.3 (RA), 234.3 > 84.3 (MPH), and 247.3 > 98.2 (mepivacaine). The dwell time for all analytes was set at 500 ms. Data were acquired and quantitation performed using Analyst software 1.2 (AB MDS Sciex).

employed. Chromatography, which will separate the analytes of interest from endogenous contaminants, is normally required. In our method, the dilution of the urine (1/100) was more than adequate to eliminate the ion suppression due to contaminants, and still allow the detection of clinically significant levels of both MPH and RA. The diluted urine

API 2000 turboionspray source. Long runs of 100 or more samples did not dirty the instrument; no loss of sensitivity was observed. The simplicity and ease of sample preparation by simple dilution allows relatively large runs to be prepared with little analyst’s time. Run times of 1.6 min per sample allowed processing of one 96-well plate in about 4 h (includ- ing autosampler delays). The presence of either RA and/or MPH in urine indicates the use of MPH. Because MPH is a relative newcomer to the list of popular drugs of abuse and because its use is less widespread, there are no specific guidelines or widely accepted urinary cut-off levels established. We developed our cut-off levels mostly based on assay reliability, but as well, we took into account the levels that would be expected after different patterns of use. Urinary MPH levels following

GC/MS The GC/MS analysis was performed using a bench top Finnigan (Fisons) MD 800 quadrupole GC/MS (Manches- ter, UK). The instrument was operated in EI+, SIR mode with dwell settings of 0.08 s and a span of F 0.10 amu. The following m/z’s were monitored: MPH—180*, 181, 208, 372; RA—180*, 181, 150; Levallorphan (IS)—176*, 272, 355 (* Indicates quantitation ion). Chromatographic separation was performed on a J&W 15 m, 0.25 id, 0.25 Am, DB-17 ms column (Chromato- graphic Specialities Inc., Canada). A GLC temperature program was used which included an initial temperature of 120 j C, a 1-min hold time, a 10 j C/min ramp until 200 j C, a 25 j C/min ramp until 280 j C, and a final hold time of

a single 25-mg oral dose were reported to range from 460 to

4020 nM in five adults [1]. MPH concentrations ranging from 3.4 AM to >100 A M were found in the urine of six arrested drivers who exhibited symptoms of hyperactivity or drowsiness [1] . This relatively large range in expected concentrations can at least partially be explained by the fact that these levels have not been correlated to creatinine concentration. It is common practice today to express ran- dom urine analytes as a ratio to creatinine concentration. Our patient population is 99% adult drug abusers, so that a specific correlation to dosage is not practical. With this information in mind, we expected concentrations significant-

ly higher than our established cut-off levels in clients who are actively abusing the drug. As well, the fact that we can

J. Eichhorst et al. / Clinical Biochemistry 37 (2004) 175–183


et al. / Clinical Biochemistry 37 (2004) 175–183 179 Fig. 3. LC/MS/MS – SRM of MPH,

Fig. 3. LC/MS/MS – SRM of MPH, RA, as well as the internal standard in a urine specimen positive for MPH abuse.

detect RA which has a longer half-life (4 h compared to 2.1 h for MPH) [1], and has been reported to be 60– 80 times more prevalent in urine than MPH, gives us a reasonable window of detection in cases of abuse (Figs. 4– 6) . We did analyze a group of 262 urine samples by both ELISA and LC/MS/MS and determined that ELISA

reported 13 specimens (5%) as false negatives. It should be noted that 9 of the 13 false negatives contained only RA and because the ELISA method detects only parent MPH, the actual false-negative rate is only 1.5%. For detection of much lower levels associated with concerns regarding compliance in children, a simple

with concerns regarding compliance in children, a simple Fig. 4. SRM LC/MS/MS chromatogram showing data for

Fig. 4. SRM LC/MS/MS chromatogram showing data for the analysis of level 2 control.


J. Eichhorst et al. / Clinical Biochemistry 37 (2004) 175–183

Eichhorst et al. / Clinical Biochemistry 37 (2004) 175–183 Fig. 5. SRM LC/MS/MS chromatogram showing data

Fig. 5. SRM LC/MS/MS chromatogram showing data for analysis of low cut-off control for RA.

solvent extraction (or SPE) can be employed which gives us a concentrating effect providing a detection limit of less than 0.5 nM (Fig. 7) . Plasma concentrations in children given a 10 –15 mg oral dose range from 17 to 107 nM for MPH and 500 –1125 nM for RA, 3– 6 h after ingestion.


Five-point calibration curves were constructed for each 96-well plate using a linear regression based on concen- tration versus peak area ratio of analyte to internal standard. A relatively small dynamic range was chosen

standard. A relatively small dynamic range was chosen Fig. 6. SRM LC/MS/MS chromatogram showing data for

Fig. 6. SRM LC/MS/MS chromatogram showing data for analysis of low cut-off control of MPH.

J. Eichhorst et al. / Clinical Biochemistry 37 (2004) 175–183


et al. / Clinical Biochemistry 37 (2004) 175–183 181 Fig. 7. A representative SRM chromatogram showing

Fig. 7. A representative SRM chromatogram showing a 50-nM standard spiked into 0.5 ml serum and extracted using chlorobutane. The reconstitution vol ume is 100 Al, which yields a concentration factor of 5. This simple extraction process eliminates most of the matrix effect and allows much lower limits of detection.

because in urine drugs of abuse screening, there is little or no consequence as to how high the urine levels are above the established cut-off value. The internal standard chosen was mepivacaine; however, it is the objective of this laboratory to obtain isotopically labelled standards for each species. Ritalin: Standards—Blank, 100, 200, 500, and 1000 nM; Linear regression (no weighting) y = 0.000246 x + 0.000258 ( r = 0.9994). RA: Standards—Blank, 1000, 2000, 4000, and 8000 nM; Linear regression (no weighting) y = 0.0000122 x + 0.00008 ( r = 0.9996).

Precision and accuracy

The intra-assay precision was determined at three levels for each analyte. Levels chosen were the cut-off level as well as 50% of the cut-off, and a higher level more reflective of actual positive urine specimens ( n = 25).


Concentration (nM)





CV = 14.9% CV = 6.9% CV = 2.7% CV = 19.4% CV = 13.3% CV = 5.5%

















Inter-assay precision was established over three runs approximately 1 week apart at the respective cut-off levels for both MPH and RA. For the MPH cut-off of 200 nM, the following results were obtained: %CV = 10.5, Accuracy = 98.2% ( n = 27). For the RA cut-off of 1000 nM, the following results were obtained: %CV = 15.3%, Accuracy = 85.5% ( n = 27). Results remained consistent over the entire 4-hour sam- pling time of one 96-well plate.


A total of 27 positive and negative urine specimens obtained from drug-dependent clients were spiked with both MPH and RA at or near the cut-off levels, and at a higher level. All spiked specimens were assayed both before spiking and after. The recovered amounts were compared to theoretical values based on amount added. The results were as follows:



Mean recovery


spiked (nM)




CV < 20% CV < 20% CV < 20% CV < 20%











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Discussion and conclusion

The above-described method was developed to provide a rapid, reliable, selective, and cost-effective method for detecting MPH abuse among drug users. With minor mod- ifications in sample preparation, it can also be used to provide very sensitive detection of MPH and its major metabolite RA in plasma to enable physicians to monitor compliance. Both screening and confirmation are accom- plished in one step using LC/MS/MS. The method is far superior to the above-mentioned ELISA method and GC/ MS method for several reasons.


The precision and accuracy of this method are compa- rable to the GC/MS method; however, it far exceeded that of the ELISA method. ELISA specificity yielded a false- positive rate (compared to GC/MS) of approximately 10%. This equates to 30 false positives/week if our workload is 300 samples/week. Even with the described extraction clean-up step, the false-positive rate (compared to GC/ MS) was only reduced to 5%. This would still provide our laboratory with approximately 15 false positives/week. Precision using the ELISA method was poor, so all samples had to be run in duplicate. This added a signif- icant cost to the analysis. Because negative results in drugs of abuse screening do not have dire consequences, we have little data on false negatives by ELISA. The LC/MS/ MS detection limits are similar to GC/MS–SIR analysis, but because of the absence of both extraction and deriv- atization steps, it provides better precision. We also en- countered much better linearity when compared to GC/MS and ELISA. Because there is no commercially available QC material specifically for detecting Ritalin as an abused drug, we have produced ‘‘in-house’’ controls at two levels for both MPH and RA. These are prepared by spiking drug-free urine with pure standard. Three sets of both control levels are run in each 96-well plate. QC results are plotted on Levy-Jennings charts and monitored as part of our laboratory QC program. The CV of these controls was <15% when analyzed repeatedly along with each run.


The fact that the tandem mass spectrometry method requires very simple and easily performed sample prepara- tion, it is far preferable to either of the other methodologies. A 96-well plate containing approximately 80 separate speci- mens can be prepared for analysis by one individual in about 0.5 h. The instrument time is about 4 h; however; this can be performed (after run set-up) with absolutely no involvement by the analyst and can be left to run reliably overnight. The sample preparation time for an equivalent

number of samples by GC/MS would be about 8– 10 h. Add to this an estimated instrument run time of 32 h (20 min/ injection) and the total time excluding data interpretation would be more than 40 h. This definitely excludes the GC/ MS procedure as a suitable high-throughput methodology. The ELISA method, due to the necessity of a sample clean up step, required about 5 h to prepare a 96-well plate. However, because the precision is such that samples had to be run in duplicate, two 96-well plates would be required for the same number of specimens and thus 10 h of time would be required. If we add 20 min of plate reading time, the total time required (excluding interpretation) to analyze 80 speci- mens would be greater than 10 h.


The initial expense of a tandem mass spectrometer is very significant. Acquisition of such an instrument may not be justified for this one assay alone; however, because of its virtually unlimited range of applications, it can be utilized for the analysis of a wide variety of different compounds. Our clinical laboratory already has an opera- tional need for this type of instrumentation in the Toxi- cology, Endocrinology, Neonatal screening, and Chemistry departments. Consideration may be given to multiple applications and shared usage to justify initial cost. Even after these considerations, a case may be made for cost recovery based on the following scenario. Reagents for our previous high-throughput ELISA method cost approxi- mately Can$3.08/test (done in duplicate). Our total staff cost/test was approximately Can$4.00/test. Including con- sumables, our yearly cost (based on a volume of 200 specimens/week and not including other overhead) would be approximately Can$80,000. This amount would almost cover the purchase or lease of a brand new instrument spread over 4 –5 years. Because a volume of 200 samples could be theoretically run in 1 –2 days, this would leave very appreciable time for other method development and applications. As well, it would require about 1/8 –1/6th the staff time and labour. Another consideration is the availability and cost of having experienced tandem mass spectrometer operators. Even with today’s more user-friendly software, it is im- portant to have competent, knowledgeable, formally edu- cated staff to operate such instrumentation. The technical skills required for clinical applications including trouble- shooting and method development are definitely more demanding than those of operating large immunoassay analyzers. In our laboratory, we have successfully trained two or three individuals to not only operate a tandem mass spectrometer, but to troubleshoot problems as they develop and to be able to develop new methods and procedures. These individuals had an undergraduate degree in bio- chemistry/analytical chemistry as well as an aptitude for computer applications and instrument maintenance and repair.

J. Eichhorst et al. / Clinical Biochemistry 37 (2004) 175–183


The method developed for determining MPH and its metabolite RA by tandem mass spectrometry using simple dilution sample preparation has been shown to provide a reliable, cost-effective, and practical means of handling the many requests we receive for detecting MPH abuse. As well, with minor modifications, the method can also be used for monitoring compliance.


[1] Baselt RC. Disposition of toxic drugs and chemicals in man. Fifth ed. Foster City, CA: Chemical Toxicology Institute; 2000. p. 566 – 8. [2] Solanto MV. Neuropsychopharmacological mechanisms of stimulant

drug action in attention-deficit hyperactivity disorder: a review and integration [Review] [290 refs]. Behav Brain Res 1998;94:127 – 52. Ramos L, Bakhtiar R, Majumdar T, Hayes M, Tse F. Liquid chromatog-


raphy/atmospheric pressure chemical ionization tandem mass spectrom- etry enantiomeric separation of dl-threo-methylphenidate, (Ritalin) using a macrocyclic antibiotic as the chiral selector. Rapid Commun Mass Spectrom 1999;13:2054 – 62. [4] Bach GA, Henion J. Quantitative capillary electrophoresis-ion-trap mass spectrometry determination of methylphenidate in human urine. J Chromatogr B Biomed Sci Appl 1998 (October 4);707:

275 – 85. [5] Solans A, Carnicero M, De La Torre R, Segura J. Simultaneous detec- tion of methylphenidate and its main metabolite, ritalinic acid, in dop- ing control. J Chromatogr B Biomed Appl 1994;658:380 – 4. [6] Diagnostix Ltd. Single Step METHYLPHENIDATE ELISA Plate Kit; Single Step ELISA Test Kit Procedure. 16-10-2000. Mississauga, ON, Canada. Ref Type: Pamphlet.