Simultaneous quantification of tiloronoxim and tilorone in human urine by liquid chromatography-tandem mass spectrometry

A simple, sensitive and specific HPLC method with tandem mass spectrometry (HPLC/MS/MS) detection has been developed and validated for the simultaneous quantification of tiloronoxim and its major active metabolite, tilorone, in human urine. The analytes, together with metoprolol, which was employed as an internal standard (IS), were extracted with a mixture solvent of chloroform/ethyl ether (1/2, v/v). The chromatographic separation was performed on a narrow-bore reversed phase HPLC column with a gradient mobile phase of methanol/water containing 15 mM ammonium bicarbonate (pH 10.5). The API 3,000 mass spectrometer was equipped with a TurboIonSpray interface and was operated on positive-ion, multiple reaction-monitoring (MRM) mode. The mass transitions monitored were m/z 426.3-->100.0, m/z 411.3-->100.0 and m/z 268.3-->116.1 for tiloronoxim, tilorone and the IS, respectively. The assay exhibited a linear dynamic range of 1-100 ng/ml for both tiloronoxim and tilorone based on the analysis of 0.2 ml aliquots of urine. The lower limit of quantification was 1 ng/ml for both compounds. Acceptable precision and accuracies were obtained for concentrations over the standard curve ranges. Run time of 8 min for each injection made it possible to analyze a high throughput of urine samples. The assay has been successfully used to analyze human urine samples from healthy volunteers.     

A multi-component LC–MS/MS method for detection of ten plant-derived psychoactive substances in urine

A sensitive and specific LC–MS/MS method for simultaneous detection of 10 plant-derived psychoactive substances (atropine, N,N-dimethyltryptamine, ephedrine, harmaline, harmine, ibogaine, lysergic acid amide, psilocin, scopolamine and yohimbine) in urine was developed. Direct injection of urine diluted with 3 deuterated internal standards allowed for a readily accessible method suitable for application in clinical intoxication cases. Separation was achieved using reversed phase chromatography and gradient elution with a total analysis time of 14 min. Electrospray ionization was used and ions were monitored in the positive selected reaction monitoring mode. The calibration curves were linear (r² > 0.999) and the total imprecision at high (1000 μg/L) and low (50 μg/L) substance concentrations were 4.9–13.8% and 8.3–26%, respectively. Infusing the analytes post column and injecting matrix samples showed limited influence by ion suppression. The multi-component method proved to be useful for investigation of authentic cases of intoxication with plant-derived psychoactive drugs and was indicated to cover the clinically relevant concentration ranges.     

Determination of clenbuterol in bovine urine using gas chromatography–mass spectrometry following clean-up on an ion-exchange resin

A gas chromatographic–mass spectrometric method was developed for the determination of residues of clenbuterol in bovine urine. The method involves a simple cation-exchange clean-up and concentration of clenbuterol in the acidified urine, followed by ethyl acetate extraction. The analyte is determined as the di-trimethylsilyl derivative and quantitated against an internal standard of penbutolol. Using a 5-ml sample of urine, a detection limit of 0.07 ng/ml can be achieved with recoveries close to 100% for fortification levels of 0.2 and 1 ng/ml. By increasing the sample volume to 50 ml, a detection limit below 0.01 ng/ml was achievable with recovery averaging 70%. The coefficient of variation of the assay ranged from 15% at 0.01 ng/ml (50-ml sample) to 6% at 1 ng/ml (5-ml sample). It was demonstrated that the method can detect the presence of clenbuterol in bovine urine at sub-ppb (ng/ml) levels using low resolution GC–MS with electron impact (EI) ionization.     

Sensitive and rapid liquid chromatography/tandem mass spectrometric assay for the quantification of chloroquine in dog plasma

A simple, sensitive and rapid liquid chromatography/tandem mass spectrometric (LC-MS/MS) method was developed and validated for quantification of chloroquine, an antimalarial drug, in plasma using its structural analogue, piperazine bis chloroquinoline as internal standard (IS). The method is based on simple protein precipitation with methanol followed by a rapid isocratic elution with 10 mM ammonium acetate buffer/methanol (25/75, v/v, pH 4.6) on Chromolith SpeedROD RP-18e reversed phase chromatographic column and subsequent analysis by mass spectrometry in the multiple reaction monitoring mode (MRM). The precursor to product ion transitions of m/z 320.3-->247.2 and m/z 409.1-->205.2 were used to measure the analyte and the IS, respectively. The assay exhibited a linear dynamic range of 2.0-489.1 ng/mL for chloroquine in dog plasma. The limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.4 and 2.0 ng/mL, respectively in 0.05 mL plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range of 2.0-489.1 ng/mL. A run time of 2.0 min for a sample made it possible to achieve a throughput of more than 400 plasma samples analyzed per day. The validated method was successfully used to analyze samples of dog plasma during non-clinical study of chloroquine.     

A capillary liquid chromatographic/tandem mass spectrometric method for the quantification of gamma-aminobutyric acid in human plasma and cerebrospinal fluid

A sensitive and reliable method for the determination of gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter, in human plasma and cerebrospinal fluid (CSF) has been developed. The method is based on capillary liquid chromatography (LC)/tandem mass spectrometry (MS/MS) using deuterium-labeled GABA (gamma-aminobutyric acid-2,2-D(2), GABA-d(2)) as internal standard. Pre-column derivatization with 7-fluoro-4-nitrobenzoxadiazole (NBD-F) was deployed, allowing both effective in-line pre-concentration and sensitive tandem MS detection of the analyte. An extraction column (10 mm x 0.25 mm, 7 microm, C(18)) was used for preconcentrating and stacking the sample. Separation was carried out on an analytical column (50 mm x 0.25 mm, 5 microm, C(18)). Characteristic precursor-to-product ion transitions, m/z 267--> 249 (for NBD-GABA) and m/z 269--> 251 (for NBD-GABA-d(2)) were monitored for the quantification. A linear calibration curve from 10 to 250 ng/mL GABA with an r(2) value of 0.9994 was obtained. Detection limit was estimated to be 5.00 ng/mL GABA (S/N = 3). Human plasma and CSF samples were analyzed. The concentrations of GABA were found to be 98.6 +/- 33.9 ng/mL (mean +/- S.D., n = 12), and 44.3 +/- 10.0 ng/mL (n = 6) in plasma and CSF, respectively.     

Determination of muscarine in human urine by electrospray liquid chromatographic-mass spectrometric

A liquid chromatography-mass spectrometry based method for determination of muscarine in human urine was developed and validated. The method involved a solid phase extraction of muscarine from urine using Strata X-CW column. Separation of muscarine was achieved within 16.0 min on a reversed phase Gemini C18 analytical column (150 mm × 2.0mm i.d., 5 μm) with a mobile phase consisted of aqueous 8 mmol/L heptafluorobutyric acid and acetonitrile in a gradient mode. Mass spectrometric detection was performed at m/z 174 and m/z 216 for muscarine and acetylmuscarine (internal standard), respectively. The linearity was satisfactory with a coefficient of determination (R(2)) 0.9993 at concentration range from 0.3 ng/mL to 2.0 μg/mL, LOD and LOQ for muscarine was 0.09 ng/mL and 0.3 ng/mL, respectively. The found out recoveries of muscarine were 96% or 95% for concentration 0.3 ng/mL and 0.2 μg/mL or 2.0 μg/mL, respectively. The precision in the intra-assay-study varied from 0.48% to 1.39% and in the inter-assay-study from 2.39% to 5.49%. The accuracy ranged from -3.3% to -6%. The validation results demonstrated that the method fulfilled satisfactory requirements for precision and accuracy across the calibration curve. The applicability of the method has been demonstrated by analyzing clinical urine samples. The method offers the fast objective identification of intoxication by muscarine and can become a routine screening alternative to more difficult microscopic examination of spores in the gastric content in clinical practice.     

High-performance liquid-chromatographic determination of warfarin enantiomers in plasma with automated on-line sample enrichment

A sensitive and selective chiral high performance liquid chromatographic method was developed for the direct determination of R- and S-warfarin enantiomers in human plasma. The method involved direct injection of human plasma onto a semipermeable surface (SPS) guard column, washing the proteins from the column with aqueous acetonitrile and back flushing the analytes onto a reversed phase ovomucoid silica HPLC column using switching valves. After separation, the analytes were simultaneously detected and quantitated with a fluorometer. The recoveries of R-warfarin from human plasma at 25 and 2500 ng/ml were 98.9% and 88.1%, respectively. The recoveries of S-warfarin at 25 and 2500 ng/ml were 105.4% and 93.9%, respectively. Using 100 microl of human plasma, the lower limit of quantification for both R- and S-warfarins was 25 ng/ml. Linear responses in analyte/internal standard peak height ratios were observed for analyte concentrations ranging from 25 to 2500 ng/ml for both enantiomers. Fluorescence chromatograms of drug-free human plasma showed no interfering peaks with retention times similar to those for R- and S-warfarins and the internal standard. Results from a 3-day validation study for both enantiomers demonstrated excellent precision (1.7-9.0%) and accuracy (97-109%) across the calibration range.     

Determination of atomoxetine in human plasma by a high performance liquid chromatographic method with ultraviolet detection using liquid-liquid extraction

A HPLC method with UV detection (210 nm) was developed and validated for the quantification of atomoxetine, a new medication for the treatment of attention deficit/hyperactivity disorder, in human plasma. Following a two-step liquid-liquid extraction with diethyl ether, the analyte and internal standard (maprotiline) were separated using an isocratic mobile phase of acetonitrile/phosphate buffer (39/61, v/v, pH 6.6) on a reverse phase Inertsil C(18) column. Linearity was verified over the range of 3.12-200 ng/mL atomoxetine in plasma. The lowest limit of detection is 2.5 ng/mL (S/N=10). This HPLC method was validated with within- and between-batch precisions of 4.9-14.4% and 4.7-13.1%, respectively. The within- and between-batch biases were -1.9 to 1.4% and 0.1-13.8%, respectively. Commonly used psychotropic drugs and frequently coadministered drugs did not interfere with the drug and internal standard. This method is simple, economical and specific, and has been used successfully in a pharmacokinetic study of atomoxetine.     

Determination of thiopental in urine sample with high-performance liquid chromatography using iodine-azide reaction as a postcolumn detection system

The reaction between iodine and azide ions induced by thiopental was utilized as a postcolumn reaction for chromatographic determination of thiopental. The method is based on the separation of thiopental on an Nova-Pak CN HP column with an acetonitrile-aqueous solution of sodium azide as a mobile phase, followed by spectrophotometric measurement of the residual iodine (lambda=350 nm) from the postcolumn iodine-azide reaction induced by thiopental after mixing an iodine solution containing iodide ions with the column effluent containing azide ions and thiopental. Chromatograms obtained for thiopental showed negative peaks as a result of the decrease in background absorbance. The detection limit (defined as S/N=3) was 20 nM (0.4 pmol injected amount) for thiopental. Calibration graphs, plotted as peak area versus concentrations, were linear from 40 nM. The elaborated method was applied to determine thiopental in urine samples. The detection limit (defined as S/N=3) was 0.025 nmol/ml urine. Calibration graphs, plotted as peak area versus concentrations, were linear from 0.05 nmol/ml urine. Authentic urine samples were analyzed, thiopental was determined at nmol/ml urine level.     

Determination of sumatriptan succinate in human plasma by high-performance liquid chromatography with coulometric detection and utilization of solid-phase extraction

Sumatriptan succinate (the analyte) and naloxone (the internal standard) were extracted from plasma with a solid-phase extraction technique. Chromatography and detection were performed by isocratic reversed-phase high-performance liquid chromatography with coulometric end-point detection. The standard curve was linear over the range 0–100 ng/ml of sumatriptan succinate in plasma. The reproducibility (as defined by the coefficient of variation, C.V.) over the range of the standard curve was 4.9–7.3%. The recovery averaged 83%. The sensitivity was 0.25 ng of sumatriptan on column (allowing a concentration of 0.5 ng/ml to be determined from a 1-ml plasma sample volume). Plasma profiles of the analyte following subcutaneous (s.c.) administration in eight normal male volunteers, are presented.     

Determination of famotidine in human plasma and urine by high-performance liquid chromatography

An improved, rapid and specific high-performance liquid chromatographic assay was developed for the determination of famotidine in human plasma and urine. Plasma samples were alkalinized and the analyte and internal standard (cimetidine) extracted with water-saturated ethyl acetate. The extracts were reconstituted in mobile phase, and injected onto a C₁₈ reversed-phase column; UV detection was set at 267 nm. Urine samples were diluted with nine volumes of a mobile phase-internal standard mixture prior to injection. The lower limits of quantification in plasma and urine were 75 ng/ml and 1.0 μg/ml, respectively; intra- and inter-day coefficients of variation were ≤10.5%. This method is currently being used to support renal function studies assessing the use of intravenously administered famotidine to characterize cationic tubular secretion in man.     

Quantitation of tadalafil in human plasma by liquid chromatography-tandem mass spectrometry with electrospray ionization

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantitation of tadalafil (I) in human plasma, a new selective, reversible phosphodiesterase 5 inhibitor. The analyte and internal standard (sildenafil, II) were extracted by liquid-liquid extraction with diethyl ether/dichloromethane (70/30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on reverse phase Xterra MS C18 column with a mobile phase of 10mM ammonium formate/acetonitrile (10/90, v/v, pH adjusted to 3.0 with formic acid). The protonate of analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 390.4 --> 268.0 and m/z 475.5 --> 58.3 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 10-1000 ng/mL for tadalafil in human plasma. The lower limit of quantitation was 10 ng/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 1.2 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Determination of MDMA and MDA in rat urine by semi-micro column HPLC-fluorescence detection with DBD-F and their monitoring after MDMA administration to rat.

A simultaneous semi-micro column HPLC method with fluorescence detection of abused drugs, such as 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), amphetamine (AP) and methamphetamine (MP) in rat urine was examined by using 4-(N,N-dimethylaminosulphonyl)-7-fluoro-1,2,3-benzoxadiazole (DBD-F) as a labelling reagent and alpha-phenylethylamine as an internal standard (IS). A sample (50 microL) of rat urine was added to 5 microL IS and 100 microL 100 mmol/L borate buffer (pH 12) and extracted with 1.5 mL n-hexane. After evaporation, 50 microL 75 mmol/L borate buffer (pH 8.5) and 50 microL 20 mmol/L DBD-F in CH3CN were added to the residue and mixed well. The resultant solution was heated for 20 min at 80 degrees C and then cooled in an ice bath. A good separation of DBD-derivatives could be achieved within 45 min using a semi-micro ODS column with an eluent of CH3CN/CH3OH/10 mmol/L imidazole-HNO3 buffer (pH 7.0) (= 45:5:50, v/v/v %). The DBD derivatives were monitored at 565 nm with an excitation at 470 nm. The calibration curves showed good linearity (r = 0.997) with 0.5-15 ng/mL detection limits at a S/N ratio of 3. MDMA and MDA in rat urine could be monitored for 15 h after a single administration of MDMA to rat (2.0 mg/kg, i.p.). The concentrations for MDMA and MDA (n = 3) were 0.13-160.1 and 0.17-10.9 microg/mL, respectively.     

Determination of tetracationic zinc(II) phthalocyanine derivative RLP068 in rabbit serum by liquid chromatography-tandem mass spectrometry

The development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of the tetracationic zinc(II) phthalocyanine derivative RLP068 in rabbit serum is described. The dodecadeuterated product (RLP068-D12) was used as co-eluting internal standard. RLP068 was isolated from serum samples by solid-phase extraction using weak cationic exchange cartridges (WCX). An oxidative derivatisation was used in order to simplify the peculiar HPLC and MS behaviour of the analyte and thus increasing sensitivity. Liquid Chromatography was carried out on a Polaris C18 Ether column (50 mm x 2.0 mm) with an isocratic run of 0.5% aqueous TFA/methanol. Detection was achieved by means of a Bruker Esquire 3000+ Ion Trap Mass Spectrometer equipped with an ESI source working in positive mode. A Multiple Reaction Monitoring method following the transitions 297.1 --> 282.1 for the analyte and 300.1 --> 282.1 + 285.1 for the internal standard was used. The analytical method was validated over the concentration range 2-65 ng/mL. lower limits of detection (LLOD) and quantification (LLOQ) were respectively 1 and 2 ng/mL. The method is innovative and applicable to pharmacokinetic studies.     

Determination of dihydroergocryptine in human plasma and urine samples using on-line sample extraction-column-switching reversed-phase liquid chromatography-mass spectrometry

A rapid and sensitive assay for the determination of dihydroergocryptine (DHEC) in human plasma and urine samples with dihydroergotamine (DHET) as the internal standard was developed. The procedure employs on-line sample preparation using an extraction pre-column and an octadecylsilylsilica (ODS) analytical column. After centrifugation human plasma or urine were injected onto the pre-column, concentrated and extracted, back-flushed onto the analytical column and eluted with a binary methanol--aqueous formic acid gradient. Either determination of DHEC as well of its mono- and dihydroxy-metabolites was performed by measurement of the signal responses from MS detection in the selected reaction monitoring (SRM) mode using the transition of the respective parent ions to the common daughter ion at m/z=270.2 amu. The limit of quantitation (LOQ) for determinations of DHEC in both plasma and urine were 25 pg/ml for injected sample volumes of 400 microl. Proportionality of signal responses versus concentration was accomplished within the range of 25-1000 pg/ml. Recovery of target analyte from plasma was 99%. Mean values of the coefficients of variation (CV) for the target analyte in plasma ranged from 1.7 to 13.8% (within-day) and 5.0 to 9.1% (between-day) and accuracy from 91.7 to 102.6% for the within-day and from 95.8 to 98.8% for the between-day measurements. The corresponding values for determinations in urine were 1.7-14.5% (within-day) and 5.3-11.8% (between-day) for CV and 95.8-110.7% (within-day) and 100.1-104.6% (between-day) for accuracy.     

LC-MS/MS determination in rabbit plasma of the main photoproduct of RLP068/Cl, a cationic sensitizer proposed for photodynamic therapy (PDT) of microbial infections

The clinical development of a sensitizer for photodynamic therapy (PDT) requires the structural identification of the photoproducts and their quantification in biological fluids and tissues. We describe the LC-MS identification of the most important photoproducts of a cationic phthalocyanine sensitizer (RLP068/Cl) and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of the main photoproduct (the cationic phthalimide derivative 3-[(1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl)oxy]-N,N,N-trimethylbenzenaminium chloride) in rabbit plasma. The tri-deuterated product was used as co-eluting internal standard. The cationic photoproduct was isolated from plasma samples by protein precipitation with perchloric acid in methanol (7%, v/v). HPLC step was performed on a Phenomenex Synergi Hydro-RP column (20 mm x 2.0 mm, 2 microm particles) with a mobile phase of 0.5% (v/v) aqueous TFA/methanol (85:15, v/v). Flow rate was 0.2 mL/min and 40 microL injection were performed. Run time was 10 min. Detection was achieved by means of a Bruker Esquire 3000+ ion trap mass spectrometer equipped with an ESI source working in positive mode. A multiple reaction monitoring method following the transitions 297.1 --> 282.1 for the analyte and 300.1 --> 282.1+285.1 for the internal standard was used. The analytical method was validated over the concentration range 0.46-91.2 ng/mL and lower limits of detection (LLOD) and quantification (LLOQ) respectively of 0.2 and 0.5 ng/mL were found.     

Determination of picropodophyllin and its isomer podophyllotoxin in human serum samples with electrospray ionization of hexylamine adducts by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of the new anticancer agent picropodophyllin (AXL1717) and its isomer podophyllotoxin levels in human serum has been developed. Monitoring of hexylamine adducts rather than proton adducts was used to optimize sensitivity. The chromatography system was an Acquity BEH C18, 2.1 mm × 50 mm 1.7 μm column with gradient elution (mobile phase A: 2.5 mM hexylamine and 5 mM formic acid in Milli-Q water and mobile phase B: methanol). The retention times were 1.4 min for picropodophyllin, 1.5 min for podophyllotoxin and 1.9 min for internal standard deoxypodophyllotoxin. The isomers were base-line separated. The analytes were detected after electrospray ionization in positive mode with selected reaction monitoring (SRM) with ion transitions m/z 516→102 for picropodophyllin and podophyllotoxin and m/z 500→102 for internal standard. The sample preparation was protein precipitation with acetonitrile (1:3) containing internal standard followed by dilution of the supernatant with mobile phase A (1:1). The limit of quantification (LOQ) was 0.01 μmol/L for picropodophyllin and podophyllotoxin. The limit of detection (LOD) at 3 times the signal to noise (S/N) was estimated below 0.001 μmol/L for picropodophyllin and podophyllotoxin. The quantification range of the method was between 0.01 μmol/L and 5 μmol/L for both isomers. The accuracy was within ±15% of the theoretical value for both picropodophyllin and podophyllotoxin and inter-assay precision did not exceed ±15%, except for the 0.016 μmol/L level of podophyllotoxin, which was 18%. The selectivity of the method was verified by analysis of two different product ions for each analyte and by analysis for interference of seven different batches of blank human serum. The combined recovery and matrix effects were about 83% for picropodophyllin and podophyllotoxin. The new LC-MS/MS method showed sufficient sensitivity and selectivity for determination of picropodophyllin and its isomer podophyllotoxin levels in human serum from subjects receiving therapeutic doses of AXL1717.     

Use of an ion-pairing reagent for high-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry determination of anionic anticoagulant rodenticides in body fluids

The on-line combination of high-performance liquid chromatography with mass spectrometry (HPLC–MS) has become a powerful tool for trace analysis thanks to the developments in interface techniques. However, non-volatile salts such as ion-pairing reagents are considered to be incompatible with HPLC–MS systems; they cause drops in analyte signals because of contamination of mass analyzers and also because of blocking of the capillary transferring ions from atmospheric pressure to the vacuum manifold. In this work, a new type of ion-pairing reagent, di-n-butylammonium acetate (DBA), was evaluated for use in HPLC–MS. DBA did not cause these problems to HPLC–MS systems; a possible explanation might be that DBA decomposed to volatile compounds under APCI conditions. In addition, DBA was very useful for obtaining sharp peaks, which resulted in high sensitivity. With this ion-pairing reagent, we developed a procedure for the measurement of five (including internal standard) anticoagulant rodenticides in whole blood and urine samples by SIM detection of [M−H]⁻ ions. Calibration range, recoveries and precision of the method were examined; detection limits as low as 1–5 ng/ml blood sample or 0.5–2.5 ng/ml urine sample were achieved.     

Determination of a novel gamma-secretase inhibitor in human plasma and cerebrospinal fluid using automated 96 well solid phase extraction and liquid chromatography/tandem mass spectrometry

A method for determination of a gamma-secretase inhibitor, cis-3-[4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]propanoic acid (A), in human plasma and cerebrospinal fluid (CSF) has been developed to support the clinical investigation of compound A for its potential treatment of Alzheimer's disease. The method is based on HPLC with atmospheric pressure chemical ionization tandem mass spectrometric detection (APCI-MS/MS) in the negative ionization mode using a heated nebulizer interface. The addition of phosphoric acid at the ratio of 10-30microL per milliliter of human plasma or CSF was required during clinical sample collection to stabilize an acylglucuronide metabolite (C), which was potentially present in human plasma and CSF. Tween 20 (10% solution) was added at the ratio of 20microL per milliliter of CSF during CSF sample collection to prevent the loss of compound A during the storage of clinical samples. The compound A and its analog internal standard (B) in treated plasma or CSF were isolated from human plasma or CSF using solid phase extraction (SPE) in the 96 well format. The isolated analyte and internal standard were chromatographed on a Phenomenex Synergi Polar RP analytical column (50mmx3.0mm, 4microm), using a mobile phase consisting of 60/40 (v/v, %) acetonitrile/water at a flow-rate of 0.5mL/min. Tandem mass spectrometric detection was performed using a Sciex API 3000 tandem mass spectrometer operated in the multiple reaction monitoring (MRM) mode using precursor to product ion transitions of 441-->175 for A and 469-->175 for B, respectively. The assays were validated over the concentration range of 0.5-200ng/mL for human plasma and CSF. Replicate analyses (n=5) of spiked standards for both assays yielded a linear response with coefficients of variation less than 7% and accuracy within 5% of the nominal concentrations. In addition, the assays were automated to improve sample throughput by utilizing a Packard Multi PROBEII automated liquid handling system and a Tom-Tec Quadra 96 system. Numerous clinical studies have been supported using these assays.     

Accurate identification and quantification of 11-nor-delta(9)-tetrahydrocannabinol-9-carboxylic acid in urine drug testing: evaluation of a direct high efficiency liquid chromatographic-mass spectrometric method

A direct liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for measurement of urinary Delta(9)-tetrahydrocannabinol carboxylic acid (THCA) was developed. The method involved dilution of the urine sample with water containing (2)H(9)-deuterated analogue as internal standard, hydrolysis with ammonia, reversed phase chromatography using a Waters ultra-performance liquid chromatography (UPLC) equipment with gradient elution, negative electrospray ionization, and monitoring of two product ions in selected reaction monitoring mode. The measuring range was 2-1000 ng/mL for THCA, and the intra- and inter-assay imprecision, expressed as the coefficient of variation, was below 5%. Influence from urine matrix on ionization efficiency was noted in infusion experiments, but was compensated for by the internal standard. Comparison with established gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry methods in authentic patient samples demonstrated accuracy in both qualitative and quantitative results. A small difference in mean ratios (~15%) may be explained by the use of different hydrolysis procedures between methods. In conclusion, the high efficiency LC-MS/MS method was capable of accurately identify and quantify THCA in urine with a capacity of 14 samples per hour.