Development and validation of a LC/MS/MS method for quantifying the next generation calcineurin inhibitor, voclosporin, in human whole blood

A rapid, accurate, and reproducible liquid chromatography electrospray tandem mass spectrometry (LC/ESI-MS/MS) method was developed and validated for the therapeutic drug monitoring of voclosporin in human whole blood. Sample aliquots of 100muL were processed utilizing a protein precipitation procedure that contained a mixture of methanol, 0.2M ZnSO(4), and deuterated voclosporin internal standard. Supernatant was injected onto a Zorbax SB-C8, 2.1x12.5mm column (at 60 degrees C), and washed with water-acetonitrile, supplemented with 0.02% glacial acetic acid and 0.02mM sodium acetate, to remove poorly retained components. After washing, water-MeOH (with 0.02% glacial acetic acid and 0.02mM sodium acetate) was used to elute the voclosporin and internal standard to the Applied Biosystems/MDS-Sciex API3000 mass spectrometer for detection in multiple reaction monitoring. Analytical performance was assessed in the range of 1-200ng/ml in whole blood. This method has been used to quantify concentrations of voclosporin in whole blood from healthy volunteers participating in a pharmacokinetic study.     

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.     

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.     

Highly sensitive quantification of 7alpha-hydroxy-4-cholesten-3-one in human serum by LC-ESI-MS/MS

We describe a highly sensitive and specific method for the quantification of serum 7alpha-hydroxy-4-cholesten-3-one (C4), which has been used as a biomarker for bile acid biosynthesis. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). C4 was extracted from human serum (2-50 mul) by a salting-out procedure, derivatized into the picolinoyl ester (C4-7alpha-picolinate), and then purified using a disposable C(18) cartridge. The resulting picolinoyl ester derivative of C4 was quantified by LC-MS/MS using the electrospray ionization mode. The detection limit of the C4 picolinoyl ester was found to be 100 fg (signal-to-noise ratio = 10), which was approximately 1,000 times more sensitive than the detection limit of C4 with a conventional HPLC-ultraviolet method. The relative standard deviations between sample preparations and between measurements by our method were calculated to be 5.7% and 3.9%, respectively, by one-way layout analysis. The recovery experiments were performed using serum spiked with 20.0-60.0 ng/ml C4 and were validated by a polynomial equation. The results showed that the estimated concentration with 95% confidence limit was 23.1 +/- 2.8 ng/ml, which coincided completely with the observed X(0) +/- SD = 23.3 +/- 1.0 ng/ml with a mean recovery of 93.4%. This method provides highly reliable and reproducible results for the quantification of C4, especially in small volumes of blood samples.     

Development and validation of Lenalidomide in human plasma by LC-MS/MS

A highly sensitive and ultra-fast high performance liquid chromatography- tandem mass spectrometry (LC–MS/MS) assay is developed and validated for the quantification of Lenalidomide in human plasma. Lenalidomide is extracted from human plasma by Liquid- Liquid Extraction by Ethyl Acetate and analyzed using a reversed phase isocratic elution on a XTerra RP18, (4.6 × 50 mM, 5 µm) column. A 0.1% Formic acid: Methanol (10:90% v/v), is used as mobile phase and detection was performed by Triple quadrupole mass spectrometry LC-MS/MS using electrospray ionization in positive mode. Fluconazole is used as the internal standard. The lower limit of quantification is 9.999 ng/mL for Lenalidomide. The calibration curves are consistently accurate and precise over the concentration range of 9.999 to 1010.011 ng/mL in plasma for Lenalidomide. This novel LC–MS/MS method competes with all the regulatory requirements and shows satisfactory accuracy and precision and is sufficiently sensitive for the performance of pharmacokinetic and bioequivalence studies in humans.     

Quantification of cidofovir in human serum by LC-MS/MS for children

A new method for the quantification of cidofovir (CDV), an acyclic nucleotide analogue of cytosine with antiviral activity against a broad-spectrum of DNA viruses, in human serum, using high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has been developed. A strong anion exchange (SAX) solid-phase extraction procedure was applied for the sample preparation. The tandem mass spectrometer was tuned in the multiple reaction monitoring mode to monitor the m/z 278.1-->234.9 and the m/z 288.1-->133.1 transitions for CDV and the internal standard 9-(2-phosphonylmethoxyethyl)guanine (PMEG), respectively, using negative electrospray ionization. The MS/MS response was linear over the concentration range from 78.125 ng/ml to 10,000 ng/ml, with a lower limit of quantification of 78.125 ng/ml. The intra- and inter-day precisions (relative standard deviation (%)) for CDV were less than 7.8% and the accuracies (% of deviation from nominal level) were within +/-12.1% for quality controls. The novel LC-MS/MS method allowed a specific, sensitive and reliable determination of CDV in human serum and was applied to investigate the yet unknown pharmacokinetic properties of CDV in a paediatric cancer patient.     

An improved approach for extraction and high-performance liquid chromatography analysis of paraquat in human plasma

A simple, sensitive, reliable and economical HPLC method for quantifying paraquat concentration in human plasma has been developed, using diethyl paraquat as an internal standard. The drugs were extracted from the sample and separated on Xtimate C18 column with a mobile phase of 15% acetonitrile in 0.1M orthophosphoric acid containing SDS (150 mg/l). The pH of the mobile phase was adjusted to 3 with triethylamine and the detection wavelength was 256 nm for both paraquat and the internal standard. The average extraction recoveries were 91.9%. Good linearity (R(2)=0.9984) was observed throughout the range of 0.02-10 μg/ml in 0.5 ml plasma. The overall accuracy of this method was 97.6-107.3% and the lower limit of detection was 0.01 μg/ml. The intra- and inter-day variations were lower than 3.65% and 2.64%, respectively. We used this method to examine the paraquat concentrations of 53 patients with acute paraquat intoxication of whom 26 (49.1%) survived. In conclusion, this method was suitable for quantification of paraquat plasma concentration in toxicological samples. It was helpful in both assessing the severity of intoxication and predicting the outcome of paraquat poisoning.     

Quantitative determination of paraquat in meconium by sodium borohydride-nickel chloride chemical reduction and gas chromatography/mass spectrometry (GC/MS)

The objective of this study was to develop a procedure for the GC/MS assay of paraquat in meconium as a biomarker of fetal exposure to paraquat. The method involved a sodium borohydride-nickel chloride reduction procedure, liquid-liquid extraction of the perhydrogenated product, concentration, and GC/MS assay. The method demonstrated good overall recovery (102.56%) with %CV (inter-assay) of less than 13%, and a limit of detection of 0.0156microg/g. Analysis of meconium samples from a study population in the Philippines (n=70) showed a 2.8% prevalence of fetal exposure to paraquat.     

Development and validation of an LC/MS/MS assay for mycophenolic acid in human peripheral blood mononuclear cells

The aim was to develop a LC/MS/MS method able to quantify mycophenolic acid (MPA) in the peripheral blood mononuclear cells (PBMCs) of transplanted patients. PBMCs were isolated from blood by a density gradient separation. The chromatographic separation was carried out on a Zorbax Stable Bond CN, 150 mmx2.1 mm, and MS/MS detection was performed after positive electrospray ionisation of the protonated parent ion. The calibration range was from 0.25 to 100 ng/sample. Extraction from the cells and ionisation recoveries reached 73.5 and 37.9%, respectively. Inaccuracy was always <10% with CVs<15%. MPA was stable at room temperature in the autosampler over 48 h and at -20 degrees C over 1.5 months. Application to clinical samples taken from patients treated with mycophenolate mofetil indicated that the method is suitable for measuring intracellular MPA.     

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.     

Determination of tegaserod by LC-ESI-MS/MS and its application to a pharmacokinetic study in healthy Chinese volunteers

A simple, rapid and sensitive high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) assay for determination of tegaserod in human plasma using diazepam as internal standard (IS) was established. After adjustment to a basic pH with sodium hydroxide, plasma was extracted by ethyl acetate and separated by high performance liquid chromatography (HPLC) on a reversed-phase C18 column with a mobile phase of methanol: 5 mM ammonium acetate (75:25, v/v, adjusting the pH to 3.5 with glacial acetic acid). The quantification of target compounds was obtained by using multiple reaction monitoring (MRM) transitions; m/z 302.5, 173.2 and 285.4, 193.2 were measured in positive mode for tegaserod and internal standard (diazepam), respectively. The lower limit of quantification (LLOQ) was 0.05 ng/ml. The calibration curves were linear over the range 0.05-8.0 ng/ml (r=0.9996) for tegaserod. The mean absolute recovery of tegaserod was more than 85.56%. Intra- and inter-day variability values were less than 9.21% and 10.02%, respectively. The samples were stable for 8h under room temperature (25 degrees C, three freeze-thaw cycles in 30 days and for 30 days under -70 degrees C). After administration of a single dose of tegaserod maleate 4 mg, 6 mg and 12 mg, respectively, the area under the plasma concentration versus time curve from time 0 h to 12 h (AUC0-12) were (2.89+/-0.88), (5.32+/-1.21) and (9.38+/-3.42) ng h/ml, respectively; peak plasma concentration (Cmax) were (1.25+/-0.53), (2.21+/-0.52) and (4.34+/-1.66) ng/ml, respectively; apparent volume of distribution (Vd/F) were (6630.5+/-2057.8), (7615.2+/-2242.8) and (7163.7+/-2057.2) l, respectively; clearance rate (CL/F) were (1851.4+/-496.9), (1596.2+/-378.5) and (1894.2+/-459.3) l/h, respectively; time to Cmax (Tmax) were (1.00+/-0.21), (1.05+/-0.28) and (1.04+/-0.16) h, respectively; and elimination half-life (t1/2) were (3.11+/-0.78), (3.93+/-0.92) and (3.47+/-0.53) h, respectively; MRT were (3.74+/-0.85), (4.04+/-0.56) and (3.28+/-0.66) h, respectively. The essential pharmacokinetic parameters after oral multiple doses (6mg, b.i.d) were as follows: Cssmax, (2.72+/-0.61) ng/ml; Tmax, (1.10+/-0.25) h; Cssmin, (0.085+/-0.01) ng/ml; Cav, (0.54+/-0.12) ng/ml; DF, (4.84+/-0.86); AUCss, (6.53+/-1.5) ngh/ml. This developed and validated assay method had been successfully applied to a pharmacokinetic study after oral administration of tegaserod maleate in healthy Chinese volunteers at a single dose of 4 mg, 6 mg and 12 mg, respectively. The pharmacokinetic parameters can provide some information for clinical medication.     

Stereoselective analysis of bupropion and hydroxybupropion in human plasma and urine by LC/MS/MS

A sensitive, stereoselective assay using solid phase extraction and LC-MS-MS was developed and validated for the analysis of (R)- and (S)-bupropion and its major metabolite (R,R)- and (S,S)-hydroxybupropion in human plasma and urine. Plasma or glucuronidase-hydrolyzed urine was acidified, then extracted using a Waters Oasis MCX solid phase 96-well plate. HPLC separation used an alpha(1)-acid glycoprotein column, a gradient mobile phase of methanol and aqueous ammonium formate, and analytes were detected by electrospray ionization and multiple reaction monitoring with an API 4000 Qtrap. The assay was linear in plasma from 0.5 to 200 ng/ml and 2.5 to 1000 ng/ml in each bupropion and hydroxybupropion enantiomer, respectively. The assay was linear in urine from 5 to 2000 ng/ml and 25 to 10,000 ng/ml in each bupropion and hydroxybupropion enantiomer, respectively. Intra- and inter-day accuracy was >98% and intra- and inter-day coefficients of variations were less than 10% for all analytes and concentrations. The assay was applied to a subject dosed with racemic bupropion. The predominant enantiomers in both urine and plasma were (R)-bupropion and (R,R)-hydroxybupropion. This is the first LC-MS/MS assay to analyze the enantiomers of both bupropion and hydroxybupropion in plasma and urine.     

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.     

Simultaneous quantification of carbamate insecticides in human plasma by liquid chromatography/tandem mass spectrometry

Carbofuran (CFN), carbosulfan (CSN) and fenobucarb (FBC) are carbamate pesticides that are widely used in gardening and agriculture for the control of insects. Human poisoning due to occupational or self-poisoning exposures is also reported, so assays are required to quantify the plasma concentration of these insecticides. An LC-MS/MS method was developed and validated for the simultaneous quantification of these three carbamate insecticides in the plasma of patients with acute intentional self-poisoning. Plasma samples were pretreated by acetonitrile for protein precipitation. Chromatography was carried out on a Luna C18(2) analytical column with gradient elution using a mobile phase containing acetonitrile and water with 10mM ammonium acetate. Mass spectrometric analysis was performed by an Applied Biosystems MDS Sciex API 2000 triple quadrupole mass spectrometer coupled with electrospray ionization (ESI) source in the positive ion mode. The total run time was 7 min. The assay was validated over a concentration range from 10 to 1000 ng/ml for CSN and FBC and 20-2000 ng/ml for CFN. The precision and accuracy for both intra- and inter-day determination of all analytes were acceptable (<15%). No significant matrix effect was observed. Stability of compounds was established for short term bench and autosampler storage as well as freeze/thaw cycles. The method was effectively applied to 270 clinical samples from patients with a history of acute intentional carbamate self-poisoning.     

Sensitive liquid chromatography/tandem mass spectrometry method for the determination of the lipophilic antipsychotic drug chlorpromazine in rat plasma and brain tissue

A simple, sensitive and robust liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for quantification of chlorpromazine in rat plasma and brain tissue. Chlorpromazine was extracted from rat plasma and brain homogenate using liquid-liquid extraction. The compounds were separated on a Waters Atlantis dC-18 (30 mm x 2.1 mm i.d., 3 microm) column using a mobile phase of acetonitrile/20 mM ammonium formate (pH 4.25 adjusted with formic acid) with gradient elution. Chlorpromazine was detected in positive ion mode using multiple reaction monitoring (MRM). The method was validated and the specificity, linearity, lower limit of quantitation (LLOQ), precision, accuracy, recoveries and stability were determined. The LLOQ was 0.2 ng/ml for plasma and 0.833 ng/g for brain tissue. The method was linear over the concentration range from 0.2 to 200.0 ng/ml for plasma and from 0.833 to 833.3 ng/g for brain tissue. The correlation coefficient (R(2)) values were more than 0.998 for both plasma and brain homogenate. The precision and accuracy for intra-day and inter-day were better than 7.54%. The relative and absolute recovery was above 84.9% and matrix effects were lower than 5.6%. This validated method has been successfully used to quantify the rat plasma and brain tissue concentration of chlorpromazine after chronic treatment.     

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.     

LC/MS analysis of NAD biosynthesis using stable isotope pyridine precursors

A liquid chromatographic-electrospray ionization ion trap mass spectrometry (LC/MS) method has been developed to measure the biosynthetic incorporation of specific precursors into NAD. The stable isotope-labeled precursors tryptophan, quinolinic acid, nicotinic acid, and nicotinamide were added to the media of human liver tumor cells (SK-HEP) grown in culture. The cells were harvested, the NAD was extracted, and the ratio of labeled to unlabeled NAD was measured using the newly developed LC/MS assay. The quantity of NAD formed from each precursor relative to an internal standard (fully labeled 13C, 15N-labeled NAD prepared from baker's yeast) was measured. The detection limit (signal-to-noise ratio 5:1) of the LC/MS method was 37 fmol (25 pg) of NAD and was linear from 20.0 ng to 25 pg. All reported NAD levels were normalized relative to cellular protein measurements. At 50 microM precursor concentrations, nicotinamide was the dominant precursor and NAD levels in the cell rose well above normal levels. Other precursors were minimally incorporated. The same methods were applied to NAD biosynthesized by macrophages derived from peripheral blood monocytes. However, the NAD concentration in macrophages was about 5% of that in SK-HEP cells and the incorporation of stable isotope-labeled substrates remained below measurable levels.     

Development and validation of a LC-MS/MS method for the determination of viaminate in human plasma

A sensitive and specific method for determination of viaminate in human plasma by using high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS) was developed in this study. The plasma samples were simply deproteinated, extracted, evaporated, and then reconstituted in 200 microl of methanol prior to analysis. Chromatographic separation was carried out on a Shimadzu VP-ODS column (250 mm x 2.0 mm, 5 microm) with a mobile phase of methanol-water (95:5, v/v) at a flow rate of 0.2 ml/min. Quantification was performed in the negative-ion electrospray ionization mode by selected ion monitoring of the product ions at m/z 164 for viaminate and m/z 109 for testosterone propionate which was used as the internal standard. The corresponding parent ions were m/z 446 and m/z 345. A linear calibration curve was observed within the concentration range of 0.10-200 ng/ml. The lowest limit of quantitation (LLOQ) was 0.1 ng/ml. The extraction-efficiency at three concentrations was 100.7, 93.6, and 99.7%. Practical utility of this new LC-MS/MS method was confirmed in pilot pharmacokinetic studies in humans following oral administration.     

Inhibition of NAD biosynthesis by paraquat in Escherichia coli

Niacin significantly reduced the bacteristatic effect of 1 mM paraquat for Escherichia coli. Without niacin (an intermediate in the salvage pathway for pyridine nucleotide coenzyme biosynthesis), the NAD concentration was decreased rapidly and significantly in E. coli during paraquat poisoning. Niacin prevented the decline in NAD in paraquat-poisoned cells; quinolinate (an intermediate in de novo NAD biosynthesis prior to the entry point of niacin) did not. These data suggest that paraquat poisons the de novo pathway of pyridine nucleotide coenzyme biosynthesis. Similar consequences have been reported to result from hyperbaric oxygen poisoning of E. coli; thus, there is growing evidence for a common mechanism of toxicity for hyperoxia and paraquat.