Quantitative determination of perfluorooctanoic acid in serum and plasma by liquid chromatography tandem mass spectrometry

A selective and sensitive method for analysis of perfluorooctanoic acid (PFOA) in human serum and plasma, utilizing liquid chromatography tandem mass spectrometry (LC-MS/MS), has been developed and thoroughly validated to satisfy strict FDA guidelines for bioanalytical methods. A simple, automated sample preparation procedure, involving extraction of the target analyte with acetonitrile on protein precipitation media in a 96-well plate format was developed, allowing efficient handling of large numbers of samples. The proposed method uses the calibration standards prepared in a surrogate matrix (rabbit serum or plasma) and (13)C-labeled PFOA as the internal standard to account for matrix effects, instrument drift, and extraction efficiency. Human serum and plasma could not be used for matrix matching of calibration standards as endogenous levels of PFOA observed in the control human serum and plasma significantly exceeded the targeted lower limit of quantitation (LLOQ) of the method. Precision and accuracy of the method were demonstrated by analysis of rabbit serum and plasma control samples fortified at 0.5, 5, and 40 ng/mL PFOA and human serum and plasma fortified at 1.0, 5.0, 40 ng/mL PFOA. The LLOQ of 0.5 ng/mL PFOA was experimentally demonstrated for rabbit and human serum and plasma. Within-day precision and accuracy, short-term stability, freeze-thaw stability, equivalence of response between PFOA and APFO (the ammonium salt of PFOA), and dilution of concentrated samples were also investigated. The results of the validation experiments comply with the precision and accuracy limits defined by the FDA guidance document: "Guidance for Industry, Bioanalytical Method Validation", May 2001.     

96-Well liquid-liquid extraction liquid chromatography-tandem mass spectrometry method for the quantitative determination of ABT-578 in human blood samples

We report here a quantitative method for the analysis of ABT-578 in human whole blood samples. Sample preparation was achieved by a semi-automated 96-well format liquid-liquid extraction (LLE) method. Aluminum/polypropylene heat seal foil was used to enclose each well of the 96-well plate for the liquid-liquid extraction. A liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) method with pre-column regeneration was developed for the analysis of sample extracts. Selective reaction monitoring (SRM) of the mass transitions m/z 983-935 and m/z 931-883 was employed for the detection of ABT-578 and internal standard, respectively. The ammonium adduct ions [M + NH(4)](+) generated from electrospray ionization were monitored as the precursor ions. The assay was validated for a linear dynamic range of 0.20-200.75ng/ml. The correlation coefficient (r) was between 0.9959 and 0.9971. The intra-assay CV (%) was between 1.9 and 13.5% and the inter-assay CV (%) was between 4.7 and 11.3%. The inter-assay mean accuracy was between 86.4 and 102.5% of the theoretical concentrations.     

Simultaneous determination of a novel KDR kinase inhibitor and its N-oxide metabolite in human plasma using 96-well solid-phase extraction and liquid chromatography/tandem mass spectrometry

To support pharmacokinetic studies, a selective and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous determination of a novel KDR kinase inhibitor (1) and its active metabolite (2) in human plasma. The method is fully automated using a Packard MultiPROBE II system and a TomTec Quadra 96 liquid handling workstation to perform sample preparation and solid-phase extraction (SPE). Following the extraction on a mixed-mode SPE using Oasis MCX 96-well plate, the analytes were separated on a Aquasil C18 column (50 mm x 2.1 mm, i.d., 3 microm) with a mobile phase consisting of acetonitrile/ammonium acetate buffer (5 mM, pH 5.0) (60/40, v/v). The run time for each injection was 4.5 min with the retention times of approximately 2.0 and 2.7 min for 1 and 2 respectively, at a flow rate of 0.25 mL/min. A tandem mass spectrometric detection was conducted using multiple reaction monitoring (MRM) under the positive ion mode with a turbo ion-spray interface. The linear ranges of the calibration curves were 0.05-400 ng/mL for 1 and 0.1-400 ng/mL for 2 on a PE Sciex API 4000 LC-MS/MS system. The lower limits of quantitation (LLOQ) of the assay were 0.05 and 0.1 ng/mL for 1 and 2 respectively, when 0.4 mL of plasma was processed. Intra-day assay precision (using five standard curves prepared by spiking compounds to five lots of plasma) was less than 4.9% for 1 and less than 9.6% for 2 on each concentration. Assay accuracy was found to be 95.1-104.6% of nominal for 1 standards and 93.5-105.6% for 2 standards. QC samples were stable when kept at room temperature for 4 h, at -70 degrees C for 10 days, and after three freeze-thaw cycles. The extraction recoveries were 80%, 83% and 84% for 1 and 2 and I.S. respectively, and no significant matrix effects were observed. The method was successfully applied to plasma samples from clinical studies after oral administration of compound 1.     

Analysis of omeprazole and 5-OH omeprazole in human plasma using hydrophilic interaction chromatography with tandem mass spectrometry (HILIC-MS/MS)--eliminating evaporation and reconstitution steps in 96-well liquid/liquid extraction

Bioanalytical methods using liquid/liquid extraction (LLE) and liquid chromatography with electrospray tandem mass spectrometry (LC-MS/MS) are widely used. The organic extracts need to be evaporated and reconstituted, hampering further improvement of throughput and automation. In this study, we demonstrated a novel approach of eliminating these two steps in 96-well LLE by using hydrophilic interaction chromatography with MS/MS (HILIC-MS/MS) on silica column with high organic/low aqueous mobile phase. Omeprazole, its metabolite 5-OH omeprazole, and internal standard desoxyomeprazole, were extracted from 0.05 ml of human plasma using 0.5 ml of ethyl acetate in a 96-well plate. A portion (0.1 ml) of the ethyl acetate extract was diluted with 0.4 ml of acetonitrile and 10 microl was injected onto a Betasil silica column (50 mm x 3.0 mm, 5 microm) and detected by API 3000 and 4000 with (+) ESI. Mobile phase with linear gradient elution consists of acetonitrile, water, and formic acid (from 95:5:0.1 to 73.5:26.5:0.1 in 2 min). The flow rate was 1.5 ml/min with total run time of 2.75 min. The method was validated for a low limit of quantitation at 2.5 ng/ml for both analytes. The method was also validated for specificity, reproducibility, stability and recovery. Lack of adverse matrix effect and carry-over was also demonstrated. The inter-day precision and accuracy of the quality control samples at low, medium and high concentration levels were <4.4% relative standard deviation (R.S.D.) and 4.1% relative error (R.E.) for omeprazole, and 4.5% R.S.D. and 5.6% R.E. for 5-OH omeprazole, respectively.     

The novel sensitive and high throughput determination of cefepime in mouse plasma by SCX-LC/MS/MS method following off-line μElution 96-well solid-phase extraction to support systemic antibiotic programs

A sensitive and high throughput off-line μElution 96-well solid-phase extraction (SPE) followed by strong cation exchange (SCX) liquid chromatography with tandem mass spectrometry (LC/MS/MS) quantification for determination of cefepime has been developed and validated in mouse plasma. Using the chemical analog, ceftazidime as an internal standard (IS), the linear range of the method for the determination of cefepime in mouse plasma was 4–2048 ng/mL with the lower limit of quantitation level (LLOQ) of 4 ng/mL. The inter- and intra-assay precision and accuracy of the method were below 9.05% and ranged from 95.6 to 113%, respectively, determined by quality control (QC) samples at five concentration levels including LLOQ. After μElution SPE, 71.1% of cefepime was recovered. The application of the validated assay for the determination of cefepime in mouse pharmacokinetics (PK) samples after intravenous (IV) and subcutaneous (SC) doses was demonstrated.     

Development of a highly sensitive and selective UPLC/MS/MS method for the simultaneous determination of testosterone and 5alpha-dihydrotestosterone in human serum to support testosterone replacement therapy for hypogonadism

A highly sensitive and selective quantitative method to accurately determine testosterone (Te) and 5alpha-dihydrotestosterone (DHT) in human serum is crucial to the success of Te replacement therapy for hypogonadism. To this end we have developed and validated a semi-automated and relatively high-throughput method in a 96-well plate format using ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC/MS/MS) for the simultaneous determination of Te and DHT in human serum. Te and DHT along with the internal standards [(2)H(3)]-Te and [(2)H(3)]-DHT were extracted from 300 microL of human serum by liquid-liquid extraction using methyl tertiary-butyl ether (MTBE), followed by derivatization with 2,3-pyridinedicarboxylic anhydride and solid-phase extraction for sample clean up. A novel chemical derivatization approach using 2,3-pyridinedicarboxylic anhydride was employed to achieve the MS sensitivity and selectivity required for DHT. Baseline separation of Te and DHT derivatives from endogenous steroid derivatives was achieved using UPLC technology on a C18 stationary-phase column with 1.7 microm particle size. The validity of using double charcoal-stripped female human serum as surrogate matrix for preparation of calibration standards was demonstrated through standard addition experiments. The method was validated over the concentration ranges of 0.2-40 ng/mL for Te and 0.01-2 ng/mL for DHT. The validation and study sample analysis results show that the method is rugged, precise, accurate, and well suited to support pharmacokinetic studies where approximately 300 samples can be extracted and analyzed in 1 day.     

Quantitative determination of buagafuran in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive and selective high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of buagafuran in human plasma. The analyte was extracted from plasma samples with hexane after addition of isotopic internal standard and chromatographed on a RP-C(8) column. The mobile phase consisted of methanol-water (90:10, v/v) and the flow rate was 0.2 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reactions monitoring (MRM) mode using positive electrospray ionization (ESI). The method was validated over the concentration range of 0.5-200 ng/mL. Inter- and intra-day precision (RSD%) were all within 15% and the accuracy (RE%) was equal or lower than 9.5%. The lower limit of quantitation (LLOQ) was 0.5 ng/mL. The extraction recovery was on average 38.1% and the detection was not affected by the matrix. The method was successfully applied to the pharmacokinetic study of buagafuran in healthy Chinese volunteers.     

Stabilization and determination of a PPAR agonist in human urine using automated 96-well liquid-liquid extraction and liquid chromatography/tandem mass spectrometry

Two stability challenges were encountered during development of an urine assay for a proliferator-activated receptor (PPAR) agonist, I (2-{[5,7-dipropyl-3-(trifluoromethyl)-1,2-benzisoxazol-6-yl]oxy}-2-methyl propionic acid), indicated for the treatment of Type II diabetes. First, the analyte was lost in urine samples due to adsorption on container surface which is a common problem during clinical sample handling. Secondly, the acylglucuronide metabolite (III), a major metabolite of I, displayed limited stability and effected the quantitation of parent drug due to the release of I through hydrolysis. Therefore, a clinical collection procedure was carefully established to stabilize I and its acylglucuronide metabolite, III, in human urine. The metabolite was not quantitated with this method. The urine samples are treated with bovine serum albumin (BSA) equal to 1.75% of the urine volume and formic acid equal to 1% of urine volume. Compound (I) and internal standard (II) were extracted from urine with 1 mL ethyl acetate using a fully automated liquid-liquid extraction in 96-well plate format. The analytes are separated by reverse phase high-performance liquid chromatography (HPLC) with tandem mass spectrometry in multiple-reaction-monitoring (MRM) mode used for detection. The urine method has a lower limit of quantitation (LLOQ) of 0.05 ng/mL with a linearity range of 0.05-20 ng/mL using 0.05 mL of urine. The method was validated and used to assay urine clinical samples.     

Automated liquid-liquid extraction based on 96-well plate format in conjunction with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for the quantitation of methoxsalen in human plasma

A sensitive, specific and high throughput bioanalytical method using automated sample processing via 96-well plate liquid-liquid extraction and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) has been developed for the determination of methoxsalen in human plasma. Plasma samples with ketoconazole as internal standard (IS) were prepared by employing 0.2mL human plasma in ethyl acetate:dichloromethane (80:20, v/v). The chromatographic separation was achieved on a Waters Acquity UPLC BEH C18 column using isocratic mobile phase, consisting of 10mM ammonium formate and acetonitrile (60:40, v/v), at a flow rate of 0.5mL/min. The linear dynamic range was established over the concentration range 1.1-213.1ng/mL for methoxsalen. The method was rugged and rapid with a total run time of 1.5min. It was successfully applied to a pivotal bioequivalence study in 12 healthy human subjects after oral administration of 10mg extended release methoxsalen formulation under fasting condition.     

High throughput assay for the determination of lumefantrine in plasma

A high throughput bioanalytical assay for the determination of lumefantrine in plasma has been developed and validated extensively. The within-day precisions for lumefantrine were 5.2, 3.5 and 2.5% at 200, 2000 and 15000 ng/mL, respectively. The between-day precisions were 4.0, 2.8 and 3.1% at 200, 2000 and 15000 ng/mL, respectively. The lower limits of quantification (LLOQ) and the limits of detection (LOD) were 25 and 10 ng/mL, respectively using 0.250 mL plasma. The average recovery of lumefantrine was 85% and independent upon concentration. The use of 96-well plate format and short chromatographic run has increased the daily sample throughput four times. The assay is particularly suitable for large therapeutic drug monitoring studies using day 7 sampling.     

Development and validation of sensitive and selective LC–MS/MS methods for the determination of BMS-708163, a γ-secretase inhibitor,in plasma and cerebrospinal fluid using deprotonated or formate adduct ions as precursor ions

BMS-708163 is a γ-secretase inhibitor that is being developed for the treatment of Alzheimer's disease. Several LC–MS/MS methods have been developed for the determination of BMS-708163 in both plasma and cerebrospinal fluid in support of dog, rat, mouse and human studies. To support non-clinical studies, an LC–MS/MS method with a lower limit of quantitation (LLOQ) of 5 ng/mL, was developed and validated in dog, rat, and mouse plasma by using the deprotonated ion as the precursor ion. To support clinical studies, an LC–MS/MS method with LLOQ of 0.1 ng/mL, was developed and validated in human plasma by using the formate adduct as the precursor ion. Formic acid (0.01%) in water and acetonitrile was found to be the most favorable mobile phases for both deprotonated and formate adduct ions in negative electrospray ionization mode. A combination of a 3M Empore™ C18 plate for SPE and a Waters Atlantis dC18 analytical column for separation was used to achieve a highly selective solid phase extraction and chromatographic procedure from plasma without dry down and reconstitution steps. In the development of an assay for BMS-708163 in cerebral spinal fluid (CSF), significant non-specific binding of BMS-708163 was observed and resolved with pre- or post-spike of 0.2% Tween 20 into CSF samples. A dilute-and-shoot LC–MS/MS method with LLOQ of 0.1 ng/mL was developed and validated to assess BMS-708163 exposure in human CSF.     

Comparison of plasma sample purification by manual liquid–liquid extraction, automated 96-well liquid–liquid extraction and automated 96-well solid-phase extraction for analysis by high-performance liquid chromatography with tandem mass spectrometry

Three extraction procedures were developed for the quantitative determination of a carboxylic acid containing analyte (I) in human plasma by high-performance liquid chromatography (HPLC) with negative ion electrospray tandem mass spectrometry (MS–MS). The first procedure was based on the manual liquid–liquid extraction (LLE) of the acidified plasma samples with methyl tert.-butyl ether. The second procedure was based on the automation of the manual LLE procedure using 96-well collection plates and a robotic liquid handling system. The third approach was based on automated solid-phase extraction (SPE) using 96-well SPE plates and a robotic liquid handling system. A lower limit of quantitation of 50 pg/ml was achieved using all three extraction procedures. The total time required to prepare calibration curve standards, aliquot the standards and plasma samples, and process a total of 96 standards and samples by manual LLE was three-times longer than the time required for 96-well SPE or 96-well LLE (4 h, 50 min vs. 1 h, 43 min). Even more importantly, the time the bioanalyst physically spent on the 96-well LLE or 96-well SPE procedure was only a small fraction of the time spent on the manual LLE procedure (<10 min vs. 4 h, 10 min). It should be noted that the 96-well SPE procedure incorporated the two steps of evaporation of the eluates to dryness and subsequent reconstitution of the dried extract. The total time required for the 96-well SPE could be reduced by 50% if the eluates were injected directly, eliminating the drying and reconstitution steps, which is achievable when sensitivity is less of an issue.     

Quantitative analysis of thymosin alpha1 in human serum by LC-MS/MS

A high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed to measure the thymosin alpha 1 (Talpha1) concentration in human serum. Tá1 in human serum was determined by solid phase extraction and reverse phase LC-MS/MS. The high-performance liquid chromatography (HPLC) system interfaced with the MS/MS system with a Turbo Ion spray interface. Positive ion detection and multiple reaction monitoring (MRM) mode were used for this human serum quantitation. Eight different concentration standards were used to establish the detection range. Six quality control (QC) and 2 matrix blanks were checked by calibration curves performed on the same day. The lower quantitation limit was 0.5 ng/mL Talpha1 in human serum. Calibration curves were established between 0.5 to 100 ng/mL by weighted linear regression. The correlation coefficients for different days were 0.9955 or greater. Quantitation of Talpha1 by the LC-MS/MS method is fast, accurate, and precise.     

Quantitative analysis of thymosin α1 in human serum by LC-MS/MS

1 high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed to measure the thymosin alpha 1 (Tα1) concentration in human serum. Tα1 in human serum was determined by solid phase extraction and reverse phase LC-MS/MS. The high-performance liquid chromatography (HPLC) system interfaced with the MS/MS system with a Turbo Ion spray interface. Positive ion detection and multiple reaction monitoring (MRM) mode were used for this human serum quantitation. Eight different concentration standards were used to establish the detection range. Six quality control (QC) and 2 matrix blanks were checked by calibration curves performed on the same day. The lower quantitation limit was 0.5 ng/mL T α1 in human serum. Calibration curves were established between 0.5 to 100 ng/mL by weighted linear regression. The correlation coefficients for different days were 0.9955 or greater. Quantitation of Tα1 by the LC-MS/MS method is fast accurate, and precise.     

Determination of morphine and morphine glucuronides in human plasma by 96-well plate solid-phase extraction and liquid chromatography-electrospray ionization mass spectrometry

There is considerable interest in quantifying morphine and its major metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). Available assays use gas chromatography-mass spectrometry or high-performance liquid chromatography (HPLC) with single or tandem mass spectrometry, ultraviolet, electrochemical, or fluorimetric detection. Nevertheless, few methods provide adequate sensitivity for all analytes, in a single injection, with the desired rate of sample throughput. A rapid and sensitive method for quantification of morphine, M3G and M6G from human plasma using HPLC with electrospray ionization mass spectrometry was developed using a Waters Oasis MCX 96-well plate for extracting both lipophilic morphine and its hydrophilic glucuronides, C18 separation using an isocratic mobile phase (methanol, acetonitrile and formic acid), and selected ion monitoring. Recoveries of morphine, M3G and M6G, respectively, were 81, 90 and 82% at the low (2, 25 and 2 ng/ml), 80, 77 and 75% at the medium (10, 250 and 10 ng/ml), and 74, 62 and 72% at the high (100, 1000 and 100 ng/ml) quality control samples. The limit of quantitation was 0.5 ng/ml morphine and M6G, and 5 ng/ml M3G. Analytes were validated over a linear range of 0.5-200 ng/ml morphine and M6G, and 5-2000 ng/ml M3G. This assay represents an improvement over existing methods through solid phase extraction with increased sample throughput (96-well plates), use of small samples (0.5 ml), and sub-nanogram detection.     

Sensitive isotope dilution liquid chromatography/tandem mass spectrometry method for quantitative analysis of bumetanide in serum and brain tissue

We have developed and validated a simple and sensitive stable isotope dilution liquid chromatography/tandem mass spectrometric (LC-MS/MS) method for the quantification of bumetanide in human serum. Samples were prepared with a simple acetonitrile based protein precipitation. The supernatant was then analyzed directly using LC-MS/MS. Chromatographic separation was achieved on a C18 reversed phase column using a methanol and water gradient. The detection was performed in selected reaction monitoring (SRM) mode via a positive electrospray ionization (ESI) interface. The method had a lower limit of quantification (LLOQ) of 1 ng/mL, linearity up to 1250 ng/mL, intra- and inter-day precision less than 10%, and accuracy within ±10%. This method was also demonstrated to be suitable for the analysis of bumetanide in rat serum and brain tissue. Bumetanide concentrations in rat serum and brain were determined for samples collected at several intervals following intraperitoneal (i.p.) injection of bumetanide, and were used to calculate bumetanide permeability through the blood-brain barrier.     

Development and validation of a rapid 96-well format based liquid-liquid extraction and liquid chromatography-tandem mass spectrometry analysis method for ondansetron in human plasma

A rapid and sensitive LC-MS/MS method for the quantification of ondansetron was developed and validated. The plasma samples were treated by a semi-automated liquid-liquid extraction (LLE) in 1.2 mL 96-well format micro-tubes. Ondansetron and the internal standard (IS) granisetron were analyzed by combined reversed phase LC-MS/MS, with positive ion electrospray ionization, using multiple reactions monitoring (MRM). The statistical evaluation for this method reveals excellent linearity, accuracy and precision values for the range of concentrations 0.25-40.0 ng/mL. The proposed method enabled the reliable determination of ondansetron in bioequivalence studies after per os administration of a 4 or 8 mg tablet.     

A 96-well single-pot protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of muraglitazar, a novel diabetes drug, in human plasma

A 96-well single-pot protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of muraglitazar, a PPAR alpha/gamma dual agonist, in human plasma. The internal standard, a chemical analogue, was dissolved in acetonitrile containing 0.1% formic acid. The solvent system was also served as a protein precipitation reagent. Human plasma samples (0.1 mL) and the internal standard solution (0.3 mL) were added to a 96-well plate. The plate was vortexed for 1 min and centrifuged for 5 min. Then the supernatant layers were directly injected into the LC/MS/MS system. The chromatographic separation was achieved isocratically on a Phenomenox C18(2) Luna column (2 mm x 50 mm, 5 microm). The mobile phase contained 20/80 (v/v) of water and acetonitrile containing 0.1% formic acid. Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API 3000. The standard curve, which ranged from 1 to 1000 ng/mL, was fitted to a 1/x weighted quadratic regression model. This single-pot approach effectively eliminated three time consuming sample preparation steps: sample transfer, dry-down, and reconstitution before the injection, while it preserved all the benefits of the traditional protein precipitation. By properly adjusting the autosampler needle offset level, only the supernatant was injected, without disturbing the precipitated proteins in the bottom. As a result, the quality of chromatography and column life were not compromised. After more than 600 injections, there was only slightly increase of column back-pressure. The validation results demonstrated that this method was rugged and provide satisfactory precision and accuracy. The method has been successfully applied to analyze human plasma samples in support of a first-in-man study. This method has also been validated in monkey and mouse plasma for the determination of muraglitazar.     

Development and validation of a liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of D-amphetamine and diphenhydramine in beagle dog plasma and its application to a pharmacokinetic study

A new drug, quick-acting anti-motion capsule (QAAMC) composed of d-amphetamine sulfate, dimenhydrinate and ginger extraction has been studied for anti-motion-sickness use. We have developed a sensitive, specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantitative determination of d-amphetamine and diphenhydramine, the main effective components of the QAAMC, using pseudoephedrine as the internal standard. The analytes and internal standard were isolated from 200 microL plasma samples by a simple liquid-liquid extraction (LLE). Reverse-phase HPLC separation was accomplished on a Zorbax SB-C18 column (100 mm x 3.0 mm, 3.5 microm) with a mobile phase composed of methanol-water-formic acid (65:35:0.5, v/v/v) at a flow rate of 0.2 mL/min. The method had a chromatographic total run time of 5 min. A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 136.0-->91.0 (D-amphetamine), 256.0-->167.0 (diphenhydramine) and 166.1-->148.0 (IS) used for quantitation. The method was sensitive with a lower limit of quantitation (LLOQ) of 0.5 ng/mL for d-amphetamine and 1 ng/mL for diphenhydramine, with good linearity in the range 0.5-200 ng/mL for D-amphetamine and 1-500 ng/mL for diphenhydramine (r(2)> or =0.9990). All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was successfully applied to pharmacokinetic study of the QAAMC in beagle dogs.     

Determination of 1,25-dihydroxyvitamin D2 in rat serum using liquid chromatography with tandem mass spectrometry

Vitamin D therapy is widely used for the treatment of hyperparathyroidism associated with chronic renal failure in renal disease patients. The vitamin D prodrug, 1α-hydroxyvitamin D(2) (1α(OH)D(2)), is used for the treatment of the end stage renal disease patients who as a result of impaired kidney function cannot convert the naturally occurring vitamin D to the active hormonal form namely 1,25-dihydroxyvitamin D(2) (1,25(OH)(2)D(2)). The systemic circulating levels of this active form are in the pg/mL range and represent a significant bioanalytical challenge for therapeutic monitoring. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is considered the gold standard for the selective and sensitive determination of small molecule therapeutics in biological matrices. However, the reported LC-MS/MS bioanalytical assays for 1,25(OH)(2)D(2) suffer from extensive sample preparation procedures or derivatization protocols to achieve the requisite sensitivity and selectivity. In this paper, we describe an assay that employs 96-well plate solid phase extraction sample preparation combined with highly sensitive LC-MS/MS instrumentation. The utility of ultra high pressure liquid chromatography to reduce the analytical run time was also demonstrated. Employing this assay a lower limit of quantitation of 25.0 pg/mL using 300 μL sample aliquot of rat serum was achieved with linearity obtained over the range of 25.0-1000 pg/mL. Both intra-day and inter-day coefficients of variation were <15% and accuracy across the assay range was within 100±7.24%. The application of the assay was demonstrated for the analysis of 1,25(OH)(2)D(2) rat serum samples to support pharmacokinetic studies conducted at doses down to sub-microgram per kilogram of 1α(OH)D(2).