LC/MS/MS analysis of quaternary ammonium drugs and herbicides in whole blood

Quaternary ammonium drugs (atracurium, bretylium, edrophonium, ipratropium, mivacurium, neostigmine, pancuronium and rocuronium) and herbicides (difenzoquat, diquat and paraquat) in human whole blood were analysed by LC/MS/MS with positive electrospray ionisation (ESI), following extraction with Bond Elut LRC-CBA cartridges. Internal standards were benzyldimethylphenylammonium chloride monohydrate and ethyl viologen for drug and herbicide analysis, respectively. Ion-pair chromatography used heptafluorobutyric acid (15 mM)-ammonium formate (20 mM) buffer adjusted to pH 3.30 with formic acid and a linear gradient from 5 to 90% methanol run over 18 min. Recoveries ranged from 79.7 to 105.1%, detection limits were between 3.6 and 20.4 ng/ml and the intra- and inter-day precisions were less than 18.6% at a concentration of 10 ng/ml. The method was applied to a case of accidental paraquat poisoning in which the concentration of paraquat in blood was 0.64 mg/l, which is within the range associated with fatal paraquat poisoning.     

Monitoring of lopinavir and ritonavir in peripheral blood mononuclear cells, plasma, and ultrafiltrate using a selective and highly sensitive LC/MS/MS assay

For the determination of the HIV protease inhibitors lopinavir and ritonavir in human plasma, plasma ultrafiltrate, and peripheral blood mononuclear cells (PBMCs) a highly sensitive and selective method has been developed, validated, and applied to samples of a healthy volunteer. BD Vacutainer CPT and Amicon Centriplus centrifugal filter devices were used for separation of PBMCs and for ultrafiltrate generation, respectively. After liquid/liquid-extraction extracts were chromatographed isocratically within 6 min on a Jupiter Proteo column. The drugs were quantified using 2H5-saquinavir as internal standard and electrospray tandem mass spectrometry in the selected reaction monitoring mode. Limits of quantification for both analytes were 4.0 ng/mL in plasma, 0.2 ng/mL in ultrafiltrate, and 0.1 ng/cell pellet (approximately 3 x 10(6) cells) in PBMCs. The calibration ranges were linear over more than three logs with an over-all accuracy varying between 98.7% and 111.5% and an over-all precision ranging from 6.2% to 14.0% (SD batch-to-batch). After a regular oral dose of Kaletra (400 mg lopinavir, 100 mg ritonavir) analyte concentrations were detectable over a full dosing interval in plasma, ultrafiltrate, and PBMCs. The method is well suited for monitoring of free and total plasma, and intracellular lopinavir/ritonavir concentrations in samples from clinical trials.     

Quantification of free mycophenolic acid and its glucuronide metabolite in human plasma by liquid-chromatography using mass spectrometric and ultraviolet absorbance detection

The immunosuppressant drug mycophenolic acid (MPA) and its major metabolite, mycophenolic acid glucuronide (MPAG), are highly bound to albumin. An HPLC-tandem-MS (HPLC/MS/MS) and an HPLC-UV assay were developed to measure free (unbound) concentrations of MPA and MPAG, respectively. Ultrafiltrate was prepared from plasma (500 microl) by ultrafiltration at 3000 x g for 20 min (20 degrees C). Both MPA and MPAG were isolated from ultrafiltrate (100 microl) by acidification and C18 solid-phase extraction. Free MPA was measured by electrospray tandem mass spectrometry using selected reactant monitoring (MPA: m/z 338.2--> 206.9) in positive ionisation mode. Chromatography was performed on a PFPP column (50 mm x 2 mm, 5 microm). Total analysis time was 7 min. The assay was linear over the range 1-200 microg/l with a limit of quantification of 1 microg/l. The inter-day accuracy and imprecision of quality controls (7.5, 40, 150 microg/l) were 94-99% and < 7%, respectively. Free MPAG was chromatographed on a C18 Nova-Pak column (150 mm x 3.9 mm, 5 microm) using a binary gradient over 20 min. The eluent was monitored at 254 nm. The assay was linear over the range 1-50 mg/l with the limit of quantification at 2.5 mg/l. The inter-day accuracy and imprecision of quality controls (5, 20, 45 mg/l) was 101-107% and < 8% (n = 4), respectively. For both methods no interfering substances were found in ultrafiltrate from patients not receiving MPA. The methods described have a suitable dynamic linear range to facilitate the investigation of free MPA and MPAG pharmacokinetics in transplant patients. Further, this is the first reported HPLC-UV method to determine free MPAG concentrations.     

Clinical determination of 17-hydroxyprogesterone in serum by LC-MS/MS: comparison to Coat-A-Count RIA method

17alpha-Hydroxyprogesterone is a metabolic precursor of cortisol; elevated levels of 17alpha-hydroxyprogesterone are indicative of congenital adrenal hyperplasia. Traditional determination by immunoassay is plagued by poor antibody specificity, resulting in significant interferences. This study explores an LC-MS/MS method for the quantitation of 17OHP in serum. Deuterated 17alpha-hydroxyprogesterone was added as internal standard, followed by solid-phase extraction, HPLC separation with a C16-amide reverse-phase column with run time of 7 min, and quantification by MS/MS (positive electrospray ionisation) in the selected reaction monitoring mode (SRM). Transitions monitored were 331>109 for the analyte and 339>113 for the deuterated internal standard. Intra-assay precision (%R.S.D.) was 7.4% at 7 nmol/L, inter-assay precision (%R.S.D.) at 2, 7 and 27 nmol/L was 15.4, 10.0 and 7.9% and accuracy at 0.9 nmol/L was 100%. The method was linear from 0.156 to 80 nmol/L. Lower limit of quantitation was 0.2 nmol/L, providing meaningful data for patients within normal range as well as those with elevated levels.     

Differentiation of isomeric malonylated flavonoid glyconjugates in plant extracts with UPLC-ESI/MS/MS

Introduction: Glycosylation at different hydroxyl groups of flavonoids and acylation of sugar moieties are ubiquitous modifications observed in plants. These modifications give rise to simultaneous presence of numerous isomeric and isobaric compounds in tissues and extracts thereof. Objective: To develop UPLC‐MS method capable for resolution of isomeric malonylated glycoconjugates of flavonoids and recognition of structural differences. Methodology: Flavonoid glycoconjugates were extracted from leaves of blue lupin (Lupinus angustifolius L.) plants with 80% methanol. Extracts were analysed using ultraperformance liquid chromatography (UPLC) combined with tandem (quadrupole–time of flight, QToF) mass spectrometry. Results: Differentiation of malonylated glycosides of isoflavones and flavones is demonstrated in this paper. The use of UPLC‐MS/MS enabled 38 flavonoid conjugates to be distinguished, including the discrimination of five different isomers of a single 3′‐O‐methylluteolin glycoside. Additionally, pseudo MS³ experiments (CID spectra registered at high cone voltages) enabled confirmation of the aglycone structures by comparison of their spectra with those obtained from aglycone standards. Conclusions: Application of UPLC‐MS/MS allows separation and identification numerous positional isomers of malonylated glycosides of flavonoids and isoflavonoids in plant material. Provided there is strict control of the MS ionisation parameters, this method may be useful for preparation of a flavonoids spectra database, enabling the inter‐laboratory comparison of analytical results. Copyright © 2008 John Wiley & Sons, Ltd.     

MS/MS profiling of taxoids from the needles of Taxus wallichiana

Ammonium cationisation has been used for taxoid profiling of partially purified methanolic extracts of needles of Taxus wallichiana growing in different regions of the Himalayas (Kashmir, Himachal Pradesh, UP Hills, Darjeeling, Sikkim and Arunachal Pradesh) by electrospray ionisation tandem mass spectrometry (MS/MS). The MS/MS spectra of the [M + NH4]+ or [M + H]+ ions gave structurally diagnostic fragment ions which revealed information about the taxane skeleton as well as the number and nature of the substituents. The rearranged 11(15-->1)-abeo-taxanes showed a characteristic elimination of the hydroxyisopropyl group with an acetoxy/benzoyloxy group from C-9. The identification of the taxoids was achieved by comparison of the MS/MS spectra with those of authentic taxoids or was based on biogenetic grounds. The results were corroborated by liquid chromatography-MS analysis. Out of the 50 taxoids identified, 21 belonged to the rearranged class. The presence of paclitaxel in the samples from four regions was confirmed: the study also revealed the occurrence of several basic taxoids in these samples. MS/MS profiling by electrospray ionisation was shown to be a fast and reliable technique for the analysis of taxoid samples.     

Application of dried blood spots combined with HPLC-MS/MS for the quantification of acetaminophen in toxicokinetic studies

A reversed phase HPLC-MS/MS method has been developed and validated for the quantitative bioanalysis of acetaminophen in dried blood spots (DBS) prepared from small volumes (15 microL) of dog blood. Samples were extracted for analysis with methanol. Detection was by positive ion TurboIonSpray ionisation combined with selected reaction monitoring MS. The analytical concentration range was 0.1-50 microg/mL. The intra-day precision and bias values were both less than 15%. Acetaminophen was stable in DBS stored at room temperature for at least 10 days. The methodology was applied in a toxicokinetic (TK) study where the data obtained from DBS samples was physiologically comparable with results from duplicate blood samples (diluted 1:1 (v/v) with water) analysed using identical HPLC-MS/MS conditions. This work demonstrates that quantitative analysis of a drug extracted from DBS can provide high quality TK data while minimising the volume of blood withdrawn from experimental animals, to an order of magnitude lower than is current practice in the pharmaceutical industry. This is the first reported application of DBS analysis to a TK study in support of a safety assessment study. The success of this and similar, related studies has led to the intent to apply DBS technology as the recommended analytical approach for the assessment of pharmacokinetics (PK)/TK for all new oral small molecule drug candidates, which have previously demonstrated a successful bioanalytical validation.     

Development and evaluation of an efficient HPLC/MS/MS method for the simultaneous determination of pseudoephedrine and cetirizine in human plasma: application to phase-I pharmacokinetic study

A sensitive, simple and highly selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and evaluated to determine simultaneously the concentrations of pseudoephedrine and cetirizine in human plasma. The chief benefit of the present method is the minimal sample preparation, as the procedure is only one-step protein precipitation. Two drugs were separated on a C(8) column and analyzed by LC/MS/MS using positive electrospray ionisation (ESI). The method had a chromatographic run time of 12.0 min and a linear calibration curve over the concentration range of 1.0-800 ng/ml for pseudoephedrine and 1.0-400 ng/ml for cetirizine, respectively. The lower limit of quantification of the two drugs was 1.0 ng/ml, respectively. The intra- and inter-batch precisions were less than 9.7%. The method described herein has been first used to reveal the pharmacokinetic characters in healthy Chinese volunteers treated with oral administration of different dosages of cetirizine dihydrochloride and controlled-released pseudoephedrine hydrochloride compound tablet, and approached the influence of a standard meal on the extent and rate of absorption of the combination tablet.     

Validation and clinical application of a high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of 10 anti-retrovirals in human peripheral blood mononuclear cells

This paper reports the validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method that allows the quantification of 10 antiretroviral (ARV) drugs in peripheral blood mononuclear cells (PBMCs) using 6 different isotopic internal standards (IS) and its clinical application. PBMCs are isolated from blood by density gradient centrifugation and drugs are extracted with a 60% methanol (MeOH) solution containing the 6 IS. The cell extract is then injected in the HPLC system and analytes are separated on a Symmetry Shield RP18 2.1 mm × 50 mm column. The different molecules are then detected by MS/MS in electrospray positive or negative ionisation modes and data are recorded using the multiple reaction monitoring (MRM) mode. Calibration curves are constructed in the range of 0.25–125 ng/ml of cell extract by a 1/x² weighted quadratic regression. The regression coefficients obtained are always greater than 0.99 and back calculated values always comprised in the range of ±15% from their nominal concentration. Mean extraction recoveries are greater than 80% for all analytes and the method is accurate and precise with CV and bias lower than 9.4%. The lower limits of quantification (LLOQ) of the different drugs range from 0.0125 to 0.2 ng/ml of cell extract. This method was successfully applied to a cohort of 98 HIV-infected patients treated with Kaletra^® (400/100 mg of lopinavir/ritonavir (LPV/RTV) twice a day, n = 48) or with Stocrin^® (600 mg once a day, n = 50) and has been tested for cellular quantification of tipranavir (TPV) in 2 patients treated with Aptivus^® (500 mg twice a day). The patients treated by Kaletra^® showed mean cell-associated concentrations (CC) of 1819.0 and 917.2 ng/ml, for LPV and RTV, respectively. Patients treated with Stocrin^® showed mean CC of 2388.11 ng/ml while both patients under Aptivus^® showed TPV CC of 4322.7 and 1078.0 ng/ml, respectively. This method can be used to analyze ARV drug concentrations within the target tissue.     

A validated LC/MS/MS method for the quantification of pyrrole-2,3,5-tricarboxylic acid (PTCA), a eumelanin specific biomarker, in human skin punch biopsies

A novel skin tissue extraction method coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS) detection was developed and validated for the analysis of endogenous pyrrole-2,3,5-tricarboxylic acid (PTCA), a eumelanin specific biomarker, in human skin punch biopsies. The analyte is extracted from the matrix (2 mm skin punch biopsies) using a simple oxidative degradation procedure. The extract supernatants are evaporated, reconstituted in mobile phase solvent, and injected into the LC/MS/MS system without further derivatization. The chromatographic separation is achieved on a reverse phase high performance liquid chromatography (HPLC) column. The accuracy and precision of the method was determined over the concentration range of 1-1000 ng/mL PTCA from human skin extracts in three validation batch runs. Inter-assay precision (%CV) and accuracy (%R.E.) of the quality control samples were 相似文献   

Sensitive assay for determining plasma tenofovir concentrations by LC/MS/MS

An LC/MS/MS assay for the determination of tenofovir (TNF) was developed and validated for use with the EDTA anticoagulated human plasma matrix. Heparin-treated plasma and serum matrices were also validated. After addition of adefovir as an internal standard, trifluoroacetic acid was used to produce a protein-free extract. Chromatographic separation was achieved with a Polar-RP Synergi, 2.0 mm x 150 mm, reversed-phase analytical column. The mobile phase was 3% acetonitrile/1% acetic acid, aq. Detection of TNF and the internal standard was achieved by ESI MS/MS in the positive ion mode using 288/176 and 274/162 transitions, respectively. The method was linear from 10 to 750 ng/ml with a minimum quantifiable limit of 10 ng/ml when 250 microl aliquots were analyzed. The usefulness of this LC/MS/MS method to routinely monitor plasma concentrations of TNF was demonstrated along with its ability to assist in the performance of pharmacokinetic studies.     

Quantification of mycophenolate mofetil in human skin extracts using high-performance liquid chromatography–electrospray mass spectrometry

A sensitive and selective method for the quantification of mycophenolate mofetil and its active metabolite mycophenolic acid in different human skin layers after dermal administration is presented. The skin layers were separated after in vitro penetration experiments and a methanolic extraction was performed. Positive ion electrospray HPLC–MS in selected ion monitoring mode was used to quantify the substances after isocratic separation by a C₁₈ analytical column. The minimum detectable concentrations were 850 pg/ml for MMF and 1 ng/ml for MPA. The peak areas depended linearly on the concentration of both drugs over the range of 25–1000 ng/ml (r²≥0.996) with accuracy ≤9.8% and precision ≤13.2%. Total imprecision at quantification limits was 15.2% at 10 ng/ml and 16.3% at 1500 ng/ml for MMF and 15.1% at 21.0 ng/ml and 17.5% at 1300 ng/ml for MPA. This HPLC–MS method will be applicable to the profiling of MMF amounts in skin and its conversion to MPA after application of different formulations.     

A high-throughput platform for the rapid screening of vitamin D status by direct infusion-MS/MS

Vitamin D is an important fat-soluble prohormone with pleiotropic effects on human health, such as immunomodulation of the innate and adaptive immune system. There is an unmet clinical need for a rapid screening platform for 25-hydroxyvitamin D (25OH-D) determination without chromatographic separation that offers better precision and accuracy than immunoassays. Here, we introduce a high-throughput method for assessing vitamin D status from blood specimens based on direct infusion-MS/MS (DI-MS/MS) following click derivatization using 2-nitrosopyridine. We developed an optimized liquid-phase extraction protocol to minimize ion suppression when directly infusing serum or plasma extracts via a capillary electrophoresis system for quantitative determination of 25OH-D. Acceptable reproducibility (mean coefficient of variation = 10.9%, n = 412), recovery (mean = 102% at 15, 30, and 45 nmol/l), and linearity (R² > 0.998) were achieved for 25OH-D with lower detection limits (limit of detection ～1.2 nmol/l, S/N ～ 3), greater throughput (～3 min/sample), and less bias than a commercial chemiluminescence immunoassay prone to batch effects. There was mutual agreement in 25OH-D concentrations from reference blood samples measured by DI-MS/MS as compared with LC-MS/MS (mean bias = 7.8%, n = 18). We also demonstrate that this method could reduce immunoassay misclassification of vitamin D deficiency in a cohort of critically ill children (n = 30). In conclusion, DI-MS/MS offers a viable alternative to LC-MS/MS for assessment of vitamin D status in support of large-scale studies in nutritional epidemiology as well as clinical trials to rapidly screen individual patients who may benefit from vitamin D supplementation.     

Simultaneous determination of cortisol, dexamethasone, methylprednisolone, prednisone, prednisolone, mycophenolic acid and mycophenolic acid glucuronide in human plasma utilizing liquid chromatography-tandem mass spectrometry

Chronic combination immunosuppressive regimens are commonly prescribed to renal transplant recipients. To develop an assay method for pharmacokinetic studies and therapeutic drug monitoring of multiple immunosuppressives, a liquid chromatography-tandem mass spectrometry (LC/MS/MS) approach for the simultaneous analysis of several glucocorticoids, mycophenolic acid (MPA) and mycophenolic acid glucuronide (MPAG) was investigated. The resultant method utilized a gradient reverse phase separation over a Symmetry C18 column using an ammonium acetate-methanol mobile phase at pH 3.5. The analytes were detected by coupling the chromatography system via electrospray to a triple quadrupole mass spectrometer. Multiple-reaction monitoring in the negative mode ion (MH-/product) was employed selecting MPA at 319.1/190.9, MPAG at 495.1/191.0, dexamethasone at 391.0/361.0, hydrocortisone at 361.1/331.1, methylprednisolone at 373.1/343.1, prednisone at 357.1/327.2, and prednisolone at 359.1/329.1. The calibration curve concentrations ranged from 3.60 ng/mL to 50 microg/mL with the lowest limit of quantitation for corticosteroids being 3.60-7.20 ng/mL and 0.656-6.75 microg/mL for MPA and MPAG, respectively. The relative standard deviation for quality control intraday variation and interday variation was between 0.76% and 9.57% for all analytes. This assay offers a versatile, unique method for multi-analyte immunosuppressive determinations during combination immunosuppression.     

Sensitive and specific LC-ESI-MS/MS method for the determination of a styrylquinoline, BA011FZ041, a potent HIV anti-integrase agent, in rat plasma

A LC-MS/MS method was validated for the determination of BA011FZ041, a styrylquinoline derivative. After addition of BA011FZ055 as internal standard (IS), the method involved solid phase extraction (SPE), LC separation with an ether-phenyl column and quantification by MS/MS after positive ESI. The calibration curve, ranging from 1 to 500 ng/mL was fitted to a 1/x-weighted quadratic regression model. Lower limit of quantification (LLOQ) was 1 ng/mL using 100 microL of plasma. Intra- and inter-assay precision and accuracy values were within the regulatory limits. The method was successfully applied to the determination of BA011FZ041 in rat plasma and PBMCs after i.v. dosing.     

High-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of diazepam, atropine and pralidoxime in human plasma

A high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS) procedure for the simultaneous determination of diazepam from avizafone, atropine and pralidoxime in human plasma is described. Sample pretreatment consisted of protein precipitation from 100microl of plasma using acetonitrile containing the internal standard (diazepam D5). Chromatographic separation was performed on a X-Terra MS C8 column (100mmx2.1mm, i.d. 3.5microm), with a quick stepwise gradient using a formate buffer (pH 3, 2mM) and acetonitrile at a flow rate of 0.2ml/min. The triple quadrupole mass spectrometer was operated in positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over the concentration ranges of 1-500ng/ml for diazepam, 0.25-50ng/ml for atropine and 5-1000ng/ml for pralidoxime. The coefficients of variation were always <15% for both intra-day and inter-day precision for each analyte. Mean accuracies were also within +/-15%. This method has been successfully applied to a pharmacokinetic study of the three compounds after intramuscular injection of an avizafone-atropine-pralidoxime combination, in healthy subjects.     

Determination of eprosartan in human plasma and urine by LC/MS/MS

A protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of eprosartan in human plasma and urine. The solvent system also served as a protein precipitation reagent. The chromatographic separation was achieved on a CAPCELL PAK C18 column (50 mmx2.0 mm, 5 microm, Shiseido). A mobile phase was consisted of 0.5% formic acid in water and 0.5% formic acid in acetonitrile (72:28). Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API3000. The standard curves, which ranged from 5 to 2000 ng/mL in human plasma and from 0.25 to 50 microg/mL in urine, were fitted to a 1/x weighted quadratic regression model. The method proved to be accurate, specific and sensitive enough to be successfully applied to a pharmacokinetic study.     

Quantification of cyclizine and norcyclizine in human plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

A rapid and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for quantification of cyclizine and its main metabolite norcyclizine in human plasma. Samples were prepared by protein precipitation with acetonitrile and cinnarizine was used as internal standard (recovery >87%). The analytes were eluted from a C8 50 mm×2.0 mm analytical column using a linear gradient of methanol and 0.05% formic acid with a total analysis time of 4 min. Analytes were detected by MS/MS using electrospray ionisation in the positive mode with multiple reactions monitoring (MRM) of the precursor ion/product ion transitions 267.2/167.2 for cyclizine and 253.2/167.2 for norcyclizine. Matrix effects were negligible. Standard curves for cyclizine and norcyclizine were linear (r(2)≥0.996) over the range 2-200 ng/mL, with 2 ng/mL representing the lower limit of quantification. Relative standard deviations were <14% for intra- and inter-day precision and the accuracy was within ±8%. The assay was successfully applied to a clinical study.     

Validation of an electrospray ionization LC/MS/MS method for quantitative analysis of vincristine in human plasma samples

Vincristine is a natural vinca alkaloid widely used in paediatric cancer treatment. Vincristine pharmacokinetics has been already studied, but few data are available in paediatric populations. A sensitive and specific liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed for the quantification of vincristine in plasma in order to investigate pharmacokinetics in a paediatric population. Two hundred microliters of plasma was added to vinblastine, used as internal standard. Chromatographic separation was achieved on a C8 HPLC column (Phenomenex Luna 50 mm × 2.0 mm, 3.0 μm) with a mobile phase gradient at a flow rate of 0.2 ml/min. Quantification was performed using the transition of 825.4 → 765.4 (m/z) for vincristine and 811.4 → 751.4 (m/z) for vinblastine. Chromatographic separation was achieved in 8 min. The limit of quantification was 0.25 ng/ml with a precision of 10.2% and an accuracy of 99.6%. The calibration curve was linear up to 50.0 ng/ml. Intra-day precision and accuracy ranged from 6.3% to 10% and from 91.9% to 100.8%, respectively. Inter-assay precision and accuracy ranged from 3.8% to 9.7% and from 93.5% to 100.5%, respectively. No significant matrix effect was observed for vincristine. A rapid, specific and sensitive LC/MS/MS method for quantification of vincristine in human plasma was developed and is now successfully applied for pharmacokinetic studies in paediatric patients.     

Study of dried blood spots technique for the determination of dextromethorphan and its metabolite dextrorphan in human whole blood by LC–MS/MS

Dried blood spots (DBSs) technology was evaluated in an assay for the quantitation of dextromethorphan (DM) and its metabolite, dextrorphan (DT), in human whole blood using high performance liquid chromatography with tandem mass spectrometry method (LC–MS/MS). Both the parent drug and metabolite were spiked in the blood matrix and subsequently allowed to dry on a specimen collection card. The dried blood spots were removed using a manual punch and then extracted into methyl tert-butyl ether (MTBE). The organic supernatant was transferred and evaporated and the residue was reconstituted in 20% acetonitrile. The overall method recovery of DM and DT was 87.8% and 95.4%, respectively. The assay was linear over the concentration range of 0.2–200 ng/mL for both analytes. Several factors that potentially affect DBS assay quantitation were investigated, such as punch size, DBS sample punch-out location, and the volume of the blood sample pipetted on the specimen collection cards. The study determined that punch size does not affect assay quantitation accuracy. Indeed, a larger punch size increases the sensitivity due to the larger sampled blood spots. Sampling from different location on the specimen collection cards shows no significant variation for both drugs. The study also shows that acceptable results can be achieved with some variation of the sample volume, which allows a simple blood sampling procedure at the test sites. To achieve the similar lower limit of quantitation (LLOQ) as the plasma assay, several blood spots at the same concentration level were stacked together and extracted. Bioanalytical assays using the DBS technique are promising given the advantages of the method over the plasma assay.