Simultaneous quantitation of d7-nefazodone, nefazodone, d7-hydroxynefazodone, hydroxynefazodone, m-chlorophenylpiperazine and triazole-dione in human plasma by liquid chromatographic-mass spectrometry

A rapid and sensitive LC-MS assay was developed and validated for the simultaneous determination of d7-nefazodone (d7-NEF), nefazodone (NEF), d7-hydroxynefazodone (d7-OH-NEF), hydroxynefazodone (OH-NEF), m-chlorophenylpiperazine (mCPP), and triazole-dione (Dione) in human plasma using trazodone (TRZ) as the internal standard (IS). A 0.1 mL aliquot of the plasma sample was precipitated with 0.1 mL of acetonitrile and vortexed for 2 min. After centrifugation, 50 microL of supernatant was mixed with 100 microL of 10 mM ammonium formate (pH = 4.0), and a 50 microL aliquot was injected onto a BDS Hypersil C18 column at a flow rate of 0.3 mL/min. The mobile phase consisting of 10 mM ammonium formate (pH = 4) and acetonitrile, 55:45 v/v, was used in an isocratic system. The mass spectrometer was programmed to admit the protonated molecules at m/z 477.2 (d7-NEF), 493.3 (d7-OH-NEF), 197.0 (mCPP), 372.0 (IS), 470.4 (NEF), 458.0 (Dione) and 486.2 (OH-NEF). Standard curves were linear (r(2) >/= 0.994) over the concentration range of 4-1000 ng/mL for Dione and 2-500 ng/mL for all other analytes. The lowest standard concentrations were the lower limits of quantitation for each analyte. The mean predicted quality control concentrations for all analytes deviated by less than 14.3% from the corresponding nominal values; the intra-assay and inter-assay precisions of the assay for all analytes were within 10.5% relative standard deviation. All analytes including the internal standard were stable in the injection solvent at room temperature for at least 24 h. The extraction recovery of the various analytes ranged from 79.2 to 109.1%. The validated assay was applied to the analysis of clinical samples obtained from a human subject who simultaneously received d7-NEF and NEF orally.     

High performance liquid chromatographic assay of Amicar, epsilon-aminocaproic acid, in plasma and urine after pre-column derivatization with o-phthalaldehyde for fluorescence detection

A simple, reliable and highly sensitive procedure was devised for measuring the levels of Amicar in blood and urine. 100 microL of serum or urine sample was added to 10 microL of a 10% w/v zinc sulfate solution and 100 microL of methanol, as previously described (Lam et al., 1980) for the removal of proteins by precipitation. 50 microL of the supernatant was then mixed with 300 microL of 1 M borate buffer containing D-valine as the internal standard before derivatization with o-phthalaldehyde. The amino acids were then separated by a stereoselective reversed-phase system using a mobile phase containing 10% of acetonitrile in 2.5 mM Cu(II) complexes of L-proline. The chromatography is highly selective, resolving Amicar from L-valine which in turn is resolved from its unnatural D-antipode, the internal standard. The procedure including sample preparation and separation required a total of 15 min. As little as 50 ng/mL of Amicar in body fluids could be detected as the o-phthalaldehyde derivative by fluorescence.     

High performance liquid chromatographic analysis of six conjugated and unconjugated estrogens in serum.

Six estrogens-estrone, equilin, estradiol, sodium estrone sulphate, sodium equilin sulphate and sodium 17-alpha-dihydroequilin sulphate in dog serum were successfully separated and quantified by a high performance liquid chromatographic method developed in our laboratories. The mobile phase was optimized by studying the effects of an organic modifier (acetonitrile) and an ion pairing reagent (tetrabutylammonium hydroxide). The serum sample work-up procedure was designed to recover both conjugated and unconjugated estrogens. The optimum method involved acetonitrile to precipitate serum proteins/peptides and extract estrogens. Residues were reconstituted in 50% acetonitrile in water for injection. The detection limits for these six estrogens via UV detection ranged from 0.5 to 5 ng on-column with a signal-to-noise ratio (S/N) of 10 for a 20 microL injection and via fluorescence 0.1 ng on-column for 17-beta-estradiol. Validation data are included for all six estrogens.     

A Simple HPLC Method for the Determination of Trazodone Human Serum

Abstract

A simple HPLC method with minimal sample preparation and good reproducibility for the determination of trazodone in serum is described. Basified serum samples were extracted using ethyl acetate containing diazepam as the internal standard (IS). Chromatography was performed on a cyanopropylsilane column with 15 μL sample injection. The mobile phase consisted of 0.02 M ammonium phosphate, pH 7.5 : acetonitrile (70:30 v/v). The eluent was monitored at 220 nm. The serum standard curve was linear from 10.0 to 8000.0 ng/mL serum. The overall within-run quality control CV was 6.3% for five concentrations (20.0, 40.0, 100.0, 250.0 and 1000.0 ng/mL) and the overall recovery from serum was 85.4%. This method has been applied to the analysis of human serum samples.     

On-line extraction coupled with liquid chromatography tandem mass spectrometry for quantitation of pravastatin and its metabolite in human serum

A high-throughput bioanalytical method for simultaneous quantitation of pravastatin and its metabolite (M1) in human serum was developed and validated using on-line extraction following liquid chromatography tandem mass spectrometry (LC-MS/MS). The on-line extraction was accomplished by the direct injection of a 50 microL serum sample, mixed 4:1 with an aqueous internal standard solution, into one of the extraction columns with aqueous 1 mm formic acid at flow rate of 3 mL/min. The separation and analysis were achieved by back-eluting the analytes from the extraction column and the analytical column to the mass spectrometer with an isocratic mobile phase consisting of 62% aqueous 1 mm formic acid and 38% acetonitrile at a flow rate of 0.8 mL/min. The second extraction column was being equilibrated while the first column was being used for analysis, and vice versa. The standard curve range was 0.500-100 ng/mL for pravastatin and M1. The lower limit of quantitation, 0.500 ng/mL for all the analytes, was achieved when 50 microL of human serum was used. The intra- and inter-day precisions were within 7.4%, and the accuracy was between 95 and 103%. The on-line extraction was finished in 0.5 min and total analysis time was 2.5 min per sample.     

Analysis of tofisopam in human serum by column-switching semi-micro high-performance liquid chromatography and evaluation of tofisopam bioequivalency

A rapid and sensitive column-switching semi-micro HPLC method is described for the direct analysis of tofisopam in human serum. The sample (100 microL) was directly injected onto the precolumn (Capcell Pak MF Ph-1), where unretained proteins were eluted to waste. Tofisopam was then eluted into an enrichment column using 13% acetonitrile in 50 mM phosphate buffer (pH 7.0) containing 5 mM sodium octanesulfonate and subsequently into the analytical column using 43% acetonitrile in 0.1% phosphoric acid containing 5 mM sodium octanesulfonate. The detection limit (2 ng/mL), good precision (CV < or = 4.2%) and speed (total analysis time 24 min) of the present method were sufficient for drug monitoring. This method was successfully applied to a bioequivalence test of two commercial tofisopam tablets.     

Liquid chromatography/mass spectrometry for the quantitation of muraglitazar in monkey plasma

A high-performance liquid chromatographic-mass spectrometric (LC/MS) assay was developed and validated for the determination of muraglitazar, a novel alpha/gamma, dual PPAR activator, in monkey plasma. The method utilized trazodone as the internal standard (IS). The extraction scheme involved a simple protein precipitation procedure with the use of a mixture of acetonitrile and methylene chloride. Separation was carried out on a BDS Hypersil C(18) analytical column (2 x 50 mm, 3 microm) and an effective chromatographic separation of muraglitazar (3.31 min) and trazadone (2.27 min) was achieved at a ssow rate of 0.3 mL/min. The mobile phase, used in an isocratic mode, consisted of 90% A (acetonitrile: 0.1% formic acid, 50:50 v/v) and 10% B (acetonitrile: 0.1% formic acid, 95:5 v/v). Detection of muraglitazar and trazodone was by positive ion turbo-ion spray mass spectrometry in the SIM mode. The mass spectrometer was programmed to admit the protonated molecules at m/z 372.0 (IS) and m/z 517.1 (muraglitazar). The standard curve, which ranged from 2 to 500 ng/mL, was fitted to a 1/x weighted linear regression model. The between run precision and within-run precision values of the assay was within 6.2% RSD. The assay accuracy was within 10.0% of the nominal values of the range of QC samples (6.0-400 ng/mL). At the lower limit of quantitation (LLQ) of 2 ng/mL, the deviation of the predicted concentrations from the nominal value of LLQ samples (n = 6) were within +/-16.6%. Muraglitazar was stable in monkey K(3)EDTA plasma for at least three freeze-thaw cycles. The processed samples (spiked samples) were stable for 48 h in auto-sampler at 10 degrees C. The average extraction recoveries of muraglitazar and IS were 83.3 and 91.9%, respectively. The assay was applied to delineate the pharmacokinetic disposition of muraglitazar in monkeys following a single oral dose.     

Determination of bradykinin and arg-bradykinin in rat muscle tissue by microdialysis and capillary column-switching liquid chromatography with mass spectrometric detection

Quantification of bradykinin peptides in limited amounts of rat muscle tissue dialysate has been performed using a packed capillary LC-ESI-TOF-MS method. The micro dialysate samples (450 microL) with added internal standard were loaded onto a 1 mm x 5 mm loading column packed with 5 microm Kromasil C18 particles by a carrier solution of 0.1% formic acid in ACN/water (5:95, v/v) at a flow rate of 250 microL/min for online preconcentration of the analytes. Back-flushed elution onto a 150 mm x 0.5 mm Zorbax C18 column packed with 5 microm particles was conducted using a linear solvent ACN/H2O gradient containing 0.1% formic acid. (Tyr8)-bradykinin was used as an internal standard and was added to the dialysis sample prior to injection. Baseline separation of bradykinin, arg-bradykinin and (tyr8)-bradykinin was achieved within 10 min. Positive ESI was performed in the m/z range of 200-1300. The method was validated in the range 0.2-1.0 ng/mL dialysate, yielding correlation coefficients of 0.995 and 0.990 for bradykinin and arg-bradykinin, respectively. The within-assay and between-assay precisions were between 4.3-9.6% and 6.2-10.6%, respectively. Both arg-bradykinin and bradykinin were detected in dialysate from rat muscle tissue, at concentrations of 0.1 and 0.4 ng/mL for bradykinin and arg-bradykinin, respectively, confirming the presence of arg-bradykinin in rat muscles.     

Quantitative determination of the polypeptide motilin in rat plasma by externally calibrated liquid chromatography/electrospray ionization mass spectrometry

We present a method for the quantitation of motilin from rat plasma by protein precipitation and liquid chromatography/mass spectrometry (LC/MS). Using external calibration, the method was linear over the concentration range 10-1000 ng/mL with an initial sample volume of 150 microL. The LC system included a C(18) column with a 300 A pore size. A linear gradient was used with a mobile phase consisting of water and acetonitrile, each with 0.2% acetic acid and 0.02% trifluoroacetic acid. Motilin was detected with the mass spectrometer in positive ion mode monitoring the 4+ charge state at m/z 675.5. The approximated limit of detection was less than 1 ng/mL and the lower limit of quantitation (LLOQ) was 10 ng/mL. The method showed a high degree of precision and accuracy both within and between runs at five validation points, including the LLOQ.     

Direct injection of plasma to determine pseudoephedrine by high performance liquid chromatography with column switching

An HPLC method has been developed for the determination of pseudoephedrine in plasma using column switching. Preparation of the sample was simple in that only 1000 microL of water was added to 200 microL of plasma before injection. A 900 microL aliquot was injected onto the precolumn. Double distilled water was used to elute and remove proteins and polar components in the sample. The components retained on the precolumn were flushed forward onto the analytical column by the mobile phase (acetonitrile-0.2 mol/L ammonium sulphate, 10:90 v/v) with automated column switching. The limit of determination of pseudoephedrine in plasma was 12 ng/mL. The relative standard deviations of intra- and inter-assay for the determination of pseudoephedrine in plasma were 1.2-9.8% over the concentration range 1020-21.8 ng/mL. The mean recovery by on-line solid phase extraction was 94.76% (RSD = 1.1%).     

Direct determination of chlorpyrifos and its main metabolite 3,5, 6-trichloro-2-pyridinol in human serum and urine by coupled-column liquid chromatography/electrospray-tandem mass spectrometry

A rapid and sensitive automated coupled-column liquid chromatography/electrospray tandem mass spectrometry (LC/LC/ES-MS/MS) method has been developed for the quantitation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol (TCP) in both human serum and urine. Human serum was first protein precipitated with acetonitrile, while urine was directly injected into the coupled-column system. A 10 microL aliquot was then analyzed using as first separation column a Discovery C18 5 microm 50 x 2.1 mm; the fraction containing the analyte was transferred on-line to the second column consisting of a ABZ+ 5 microm 100 x 2.1 mm, which was connected to the electrospray source (Z-spray) of a Quattro LC triple-quadrupole instrument. Chlorpyrifos was detected in positive ion mode using four multi reaction monitoring (MRM) transitions while TCP was measured in negative ion mode using three pseudo-MRM transitions. The clean-up performed by the coupled-column approach avoids the use of an internal standard for the correct quantitation of both analytes, and the highly automated procedure renders a sample throughput of more than 100 samples per day. Both compounds can be determined using the same set-up, the only difference in the procedure being the composition of the first mobile phase. The method has proved to be fast, reliable and sensitive, yielding calibration curves for both analytes with correlation coefficients greater than 0.9995. The repeatability and reproducibility at 5 and 50 ng/mL was lower than 8%. The accuracy and precision were evaluated by means of recovery experiments from fortified serum (5-50 ng/mL) and urine (1-10 ng/mL) samples, obtaining satisfactory recoveries for both compounds (87-113% in serum, and 98-109% in urine), with coefficients of variation (CVs) less than 10%. The detection limits were similar for chlorpyrifos and metabolite: 1.5 ng/mL in serum, and 0.5 ng/mL in urine, where no sample handling took place. The validated procedures provide excellent tools for the specific assessment of occupational exposure to the organophosphorus pesticide chlorpyrifos, throughout the analysis of both human serum and urine, and it is more selective and sensitive than the current assay based on the measurement of the decrease in the cholinesterase activity.     

The use of liquid chromatography/mass spectrometry for quantitative analysis of oxycodone, oxymorphone and noroxycodone in Ringer solution, rat plasma and rat brain tissue

Sensitive and reproducible methods for the determination of oxycodone, oxymorphone and noroxycodone in Ringer solution, rat plasma and rat brain tissue by liquid chromatography/mass spectrometry are described. Deuterated analogs of the substances were used as internal standards. Samples in Ringer solution were analyzed by direct injection of 10 microL Ringer solution diluted by an equal volume of water. The limit of quantification was 0.5 ng/mL and the method was linear in the range of 0.5-150 ng/mL for all substances. To analyze oxycodone and oxymorphone in rat plasma, 50 microL of plasma were precipitated with acetonitrile, and the supernatant was directly injected onto the column. To analyze oxycodone, oxymorphone and noroxycodone in rat plasma, 100 microL of rat plasma were subjected to a C18 solid-phase extraction (SPE) procedure, before reconstituting in mobile phase and injection onto the column. For both methods the limit of quantification in rat plasma was 0.5 ng/mL and the methods were linear in the range of 0.5-250 ng/mL for all substances. To analyze the content of oxycodone, oxymorphone and noroxycodone in rat brain tissue, 100 microL of the brain homogenate supernatant were subjected to a C18 SPE procedure. The limit of quantification of oxycodone was 20 ng/g brain, and for oxymorphone and noroxycodone 4 ng/g brain, and the method was linear in the range of 20-1000 ng/g brain for oxycodone and 4-1000 ng/g brain for oxymorphone and noroxycodone. All methods utilized a mobile phase of 5 mM ammonium acetate in 45% acetonitrile, and a SB-CN column was used for separation. The total run time of all methods was 9 min. The intra-day precision and accuracy were <11.3% and <+/-14.9%, respectively, and the inter-day precision and accuracy were <14.9% and <+/-6.5%, respectively, for all the concentrations and matrices described.     

Determination of ribavirin in human serum and plasma by capillary electrophoresis

The electrophoretic separation of ribavirin and 5-methylcytidine (internal standard) by capillary electrophoresis was examined. Separation was achieved using reverse polarity in a 100 mM borate electrolyte, pH 9.1, with 5 mM spermine added to reduce the electroosmotic flow. Sample preparation based on acetonitrile protein precipitation was found to be unsuitable for ribavirin analysis in patient samples due to insufficient sensitivity and interferences. Solid-phase extraction employing phenyl boronic acid cartridges provided cleaner separations. Using this approach with 500 microL sample and reconstitution of the dried extract into 100 microL of 33% v/v 100 mM phosphate buffer, pH 6.4 / 67% v/v acetonitrile, the detection and quantitation limits were determined to be 0.05 and 0.10 microg/mL, respectively, a sensitivity that is suitable for therapeutic drug monitoring of ribavirin in human plasma and serum samples. The method was validated and compared to a high-performance liquid chromatography (HPLC) method, showing excellent agreement between the two for a set of samples that stemmed from patients being treated with ribavirin and interferon-alpha-2b for a hepatitis C virus infection.     

气相色谱-质谱法测定氯氮平及其去甲基代谢物

建立了测定人血清中氯氮平及其去甲基代谢物的柱前衍生化气相色谱－质谱选择离子监测的分析方法。以三氟乙酸酐作酰化剂,对衍生化条件和样品预处理方法实施了优化。氯氮平和去甲氯氮平的线性范围为１～１２８μｇ／Ｌ,最低检测浓度：氯氮平为０．１μｇ／Ｌ,去甲氯氮平为０．２μｇ／Ｌ,两者的回收率均大于８３％,相对标准偏差都小于１０％。将所建立的方法应用于服用细胞色素氧化酶Ｐ４５０１Ａ２抑制剂前后低剂量氯氮平的药代动力学自身对照试验中,结果显示氯氮平的代谢水平明显受Ｐ４５０１Ａ２活性的影响。     

Direct determination of five fluoroquinolones in chicken whole blood and in veterinary drugs by HPLC

A direct, accurate, and sensitive chromatographic analytical method for the quantitative determination of five fluoroquinolones (enoxacin, ofloxacin, norfloxacin, ciprofloxacin, and enrofloxacin) in chicken whole blood is proposed in the present study. For quantitative determination lamotrigine was used as internal standard at a concentration of 20 ng/microL. The developed method was successfully applied to the determination of enrofloxacin, as the main component of commercially available veterinary drugs. Fluoroquinolone antibiotics were separated on an Inertsil (250 x 4 mm) C8, 5 microm, analytical column, at ambient temperature. The mobile phase consisted of a mixture of citric acid (0.4 mol L(-1))-CH3OH-CH3CN (87:9:4% v/v) leading to retention times less than 14 min, at a flow rate 1.4 mL min(-1). UV detection at 275 nm provided limits of detection of 2 ng/mL per 20 microL injected volume for enoxacin, norfloxacin, and ciprofloxacin, 0.4 ng/mL for ofloxacin, and 4 ng/mL for enrofloxacin. Preparation of chicken blood samples is based on the deproteinization with acetonitrile while the pharmaceutical drug was simply diluted with water. Peaks of examined analytes in real samples were identified by means of a photodiode array detector. The method was validated in terms of within-day (n=6) precision and accuracy after chicken whole blood sample deproteinization by CH3CN. Using 50 microL of chicken blood sample, recovery rates at fortification levels of 40, 60, and 80 ng ranged from 86.7% to 103.7%. The applicability of the method was evaluated using real samples from chicken under fluoroquinolone treatment.     

Development and validation of an improved method for the quantitation of sertraline in human plasma using LC-MS-MS and its application to bioequivalence studies

A rapid and sensitive LC-MS-MS method for the quantitation of sertraline in human plasma was developed and validated. Sertraline and the internal standard, telmisartan, were cleaned up by protein precipitation from 100 μL of plasma sample, and analyzed on a TC-C18 column (5 μm, 150 × 4.6 mm i.d.) using 70% acetonitrile and 30% 10 mM ammonium acetate (0.1% formic acid) as mobile phase. The method was demonstrated to be linear from 0.1 ng/mL to 50 ng/mL with the lower limit of quantitation of 0.1 ng/mL. Intra- and inter-day precision were below 4.40% and 3.55%. Recoveries of sertraline at low, medium, and high levels were 88.0 ± 2.3%, 88.2 ± 1.9%, and 90.0 ± 2.0%, respectively. The method was successfully applied to a bioequivalence study of sertraline after a single oral administration of 50 mg sertraline hydrochloride tablets.     

Direct determination of four fluoroquinolones,enoxacin, norfloxacin,ofloxacin, and ciprofloxacin,in pharmaceuticals and blood serum by HPLC

A rapid, accurate and sensitive method has been developed for the quantitative determination of four fluoroquinolone antimicrobial agents, enoxacin, norfloxacin, ofloxacin and ciprofloxacin, with high in-vitro activity against a wide range of Gram-negative and Gram-positive organisms.A Kromasil 100 C(8) 250 mm x 4 mm, 5 microm analytical column was used with an eluting system consisting of a mixture of CH(3)CN-CH(3)OH-citric acid 0.4 mol L(-1) (7:15:78 %, v/v). Detection was performed with a variable wavelength UV-visible detector at 275 nm resulting in limits of detection: 0.02 ng per 20 microL injection for enoxacin and 0.01 ng for ofloxacin, norfloxacin and ciprofloxacin. Hydrochlorothiazide (HCT) was used as internal standard at a concentration of 2 ng microL(-1). A rectilinear relationship was observed up to 2 ng microL(-1) for enoxacin, 12 ng microL(-1) for ofloxacin, 3 ng microL(-1) for norfloxacin, and 5 ng microL(-1) for ciprofloxacin. Separation was achieved within 10 min. The statistical evaluation of the method was examined by performing intra-day (n=8) and inter-day precision assays (n=8) and was found to be satisfactory with high accuracy and precision. The method was applied to the direct determination of the four fluoroquinolones in human blood serum. Sample pretreatment involved only protein precipitation with acetonitrile. Recovery of analytes in spiked samples was 97+/-6% over the range 0.1-0.5 ng microL(-1).     

Sensitive LC-ESI/MS/MS assay for the quantification and pharmacokinetic study of roxithromycin in human serum

A simple and sensitive LC-ESI/MS/MS method is developed and evaluated to determine the concentrations of roxithromycin in human serum. Serum proteins are precipitated with methanol with clarithromycin as the internal standard. In order to reduce the pollution of sample, after vortex mixing and centrifugation, the supernatants are diluted with mobile phase before analysis on a Phenomenex Luna CN column (100 mm × 2.0mm i.d., 3 μm). The mobile phase composes of methanol, acetonitrile and 0.1% formic acid and 0.1% ammonium acetate in water (3: 3: 4, v/v/v) at a flow rate of 0.2 mL/min. The linearity ranges from 10 to 20480 ng/mL. The extraction recoveries of roxithromycin range from 97 to 101%. The method is successfully used to pharmacokinetic study of roxithromycin after an oral administration dose of 300 mg roxithromycin tablets to 20 healthy volunteers.     

On-line SPE-Nano-LC-Nanospray-MS for rapid and sensitive determination of perfluorooctanoic acid and perfluorooctane sulfonate in river water

An instrumental set up including on-line solid-phase extraction, nano-liquid chromatography, and nanospray mass spectrometry is constructed to improve the sensitivity for quantitation of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) in surface water. Sample volumes of 1000 microL are loaded onto a microbore 1.0-mm i.d. x 5 mm, 5 microm Kromasil C(18) enrichment column by a carrier solution consisting of 10mM ammonium acetate in acetonitrile-water (10:90, v/v) at a flow rate of 250 microL/min, providing on-line analyte enrichment and sample clean-up. Backflushed elution onto a 0.1-mm i.d. x 150 mm, 3.5 microm Kromasil C(18) analytical column is conducted using an acetonitrile-10mM ammonium acetate solvent gradient from 30% to 70% acetonitrile. Water samples are added with internal standard (perfluoroheptanoic acid) and filtrated prior to injection. The mass limits of detection of PFOA and PFOS are 0.5 and 1 pg, respectively, corresponding to concentration limits of detection of 500 pg/L and 1 ng/L, respectively. The total time spent on sample preparation, chromatography, and detection is approximately 12 min per sample. The method was employed for the determination of PFOS and PFOA in urban river water.     

Quantitative analysis of delta9-tetrahydrocannabinol in preserved oral fluid by liquid chromatography-tandem mass spectrometry

A rapid and sensitive method for the analysis of delta9-tetrahydrocannabinol (THC) in preserved oral fluid was developed and fully validated. Oral fluid was collected with the Intercept, a Food and Drug Administration (FDA) approved sampling device that is used on a large scale in the U.S. for workplace drug testing. The method comprised a simple liquid-liquid extraction with hexane, followed by liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis. Chromatographic separation was achieved using a XTerra MS C18 column, eluted isocratically with 1 mM ammonium formate-methanol (10:90, v/v). Selectivity of the method was achieved by a combination of retention time, and two precursor-product ion transitions. The use of the liquid-liquid extraction was demonstrated to be highly effective and led to significant decreases in the interferences present in the matrix. Validation of the method was performed using both 100 and 500 MicroL of oral fluid. The method was linear over the range investigated (0.5-100 ng/mL and 0. 1-10 ng/mL when 100 and 500 microL, respectively, of oral fluid were used) with an excellent intra-assay and inter-assay precision (relative standard deviations, RSD <6%) for quality control samples spiked at a concentration of 2.5 and 25 ng/mL and 0.5 and 2.5 ng/mL, respectively. Limits of quantification were 0.5 and 0.1 ng/mL when using 100 and 500 microL, respectively. In contrast to existing GC-MS methods, no extensive sample clean-up and time-consuming derivatisation steps were needed. The method was subsequently applied to Intercept samples collected at the roadside and collected during a controlled study with cannabis.