Direct analysis of plasma samples for drug discovery compounds using mixed-function column liquid chromatography tandem mass spectrometry

A sensitive, efficient, high throughput, direct injection bioanalytical method based on a single column and high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS) was developed for pharmacokinetic analysis of early drug discovery compounds in plasma samples. After mixing with a working solution containing an internal standard each plasma sample was directly injected into a polymer-coated mixed-function column for sample cleanup, enrichment and chromatographic separation. The stationary phase incorporates hydrophilic polyoxyethylene groups and hydrophobic groups to the polymer-coated silica. This allows proteins and macromolecules to pass through the column due to restricted access to the surface of the packing while retaining the drug molecules on the bonded hydrophobic phase. The analytes retained in the column with a largely aqueous liquid mobile phase were then chemically separated by switching to a strong organic mobile phase. The column effluent was diverted from waste to the mass spectrometer for analyte detection. Within 200 plasma sample injections the response ratio (analyte vs. internal standard, %CV = 4.6) and the retention times for analyte and internal standard were found consistent and no column deterioration was observed. The recoveries of test compound in various plasma samples were greater than 90%. The total analysis time was 相似文献   

High-speed solubility screening assay using ultra-performance liquid chromatography/mass spectrometry in drug discovery

Ultra-performance liquid chromatography (UPLC) was investigated as an alternative to high-performance liquid chromatography (HPLC) for analyzing pharmaceutical drug candidates. We previously developed a 96-well-based high-speed solubility assay system (HSSOL) using HPLC/UV and a LogD assay system (HSLogD) using HPLC/MS [Y. Dohta, T. Yamashita, S. Horiike, T. Nakamura, T. Fukami, Anal. Chem. 79 (2007) 8312]. We have introduced the UPLC/MS system into this previously developed HSSOL system to increase throughput. Results obtained by the UPLC/MS and HPLC/UV systems showed good agreement, validating the usefulness of the UPLC/MS system. A high-speed solubility assay system was developed employing the UPLC/MS system, thereby tripling the throughput.     

Quantitative determination of sparfloxacin in rat plasma by liquid chromatography/tandem mass spectrometry

Sparfloxacin, a fluoroquinolone antibiotic, is used for the treatment of bacterial infection. A quantification method using mass spectrometry was developed for the determination of sparfloxacin in rat plasma. After simple protein precipitation with acetonitrile, the analytes were chromatographed on a reversed‐phase C₁₈ column and detected by liquid chromatography/tandem mass spectrometry with electrospray ionization. The accuracy and precision of the assay were in accordance with FDA regulations for validation of bioanalytical methods. This method was applied to measure the plasma sparfloxacin concentrations after a single oral administration of sparfloxacin in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantitative high-throughput determination of endogenous retinoids in human plasma using triple-stage liquid chromatography/tandem mass spectrometry

A high-throughput ultrasensitive analytical method based on liquid chromatography with positive ion atmospheric pressure chemical ionization (APCI) coupled to tandem mass spectrometric detection (LC/MS/MS) was developed for the determination of all-trans-4-oxo-retinoic acid (at4oxoRA), 13-cis-4-oxo-retinoic acid (13c4oxoRA), 13-cis-retinoic acid (13cRA), all-trans-retinoic acid (atRA) and all-trans-retinol (atROH) in human plasma. A stable isotope of atRA was used as internal standard (IS). The analytes and IS were isolated from 100 microL plasma by acetonitrile mono-phase extraction (MPE) performed in black 96-well microtiterplates. A 100 microL injection was focused on-column and chromatographed on an Agilent ZORBAX SB-C18 rapid-resolution high-throughput (RRHT) column with 1.8-microm particles (4.6 mmx50 mm) maintained at 60 degrees C. The initial mobile phase composition was acetonitrile/water/formic acid (10:90:0.1, v/v/v) delivered at 1.8 mL/min. Elution was accomplished by a fast gradient to acetonitrile/methanol/formic acid (90:10:0.1, v/v/v). The method had a chromatographic total run time of 7 min. An Applied Biosystems 4000 Q TRAP linear tandem mass spectrometer equipped with a heated nebulizer (APCI) ionization source was operated in multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions m/z 315.4-->297 (4-oxo-retinoic acids), 301.2-->205 (retinoic acids), 305.0-->209 (IS) and 269.2-->93 (retinol) used for quantification. The assay was fully validated and found to have acceptable accuracy, precision, linearity, sensitivity and selectivity. The mean extraction recoveries from spiked plasma samples were 80-105% for the various retinoids at three different levels. The intra-day accuracy of the assay was within 8% of nominal and intra-day precision was better than 8% coefficient of variance (CV) for retinoic acids. Inter-day precision results for quality control samples run over a 12-day period alongside clinical samples showed mean precision better than 12.5% CV. The limit of quantification was in the range of 0.1-0.2 ng/mL and the mass limit of detection (mLOD) was in the range 1-4 pg on column for the retinoic acids. The assay has been successfully applied to the analysis of 1700 plasma samples.     

Determination of brain and plasma drug concentrations by liquid chromatography/tandem mass spectrometry

A method is described for the evaluation of drug concentrations in plasma and brain from treated rats. The analyte is recovered from plasma or brain homogenate by liquid-liquid extraction and subsequently analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). A simple experimental protocol renders the procedure valuable for obtaining information rapidly on brain penetration and plasma exposure of specific classes of compounds. This methodology has been applied to evaluate brain penetration with 30 different compounds from the same discovery program. In an attempt to increase throughput in our screening efforts, mixture dosing was evaluated. Results from single compound administration were compared with results following administration of a mixture of four compounds. Preliminary results, with specific classes of compounds, show no major differences (ranking order) in brain or plasma concentrations between mixture dosing and single compound administration, suggesting that mixture dosing could be applicable to brain penetration studies in the drug discovery phase.     

Quantitative analysis of docetaxel in human plasma using liquid chromatography coupled with tandem mass spectrometry

An assay for the quantitative determination of docetaxel in human plasma is described. Docetaxel was extracted from the matrix using liquid-liquid extraction with ter-butylmethylether, followed by high-performance liquid chromatographic analysis using an alkaline eluent. Paclitaxel was used as internal standard. Positive ionization electrospray tandem mass spectrometry was performed for selective and sensitive detection. The method was validated according to the FDA guidelines on bioanalytical method validation. The validated range for docetaxel was from 0.25--1000 ng/mL using 200 microL plasma aliquots. The method requires only a limited volume (200 microL) of human plasma and the method can be applied in studies requiring a low lower limit of quantitation of 0.25 ng/mL. The assay was applied successfully in several clinical and pharmacological studies with docetaxel.     

Quantitative analysis of acetaminophen and its six metabolites in rat plasma using liquid chromatography/tandem mass spectrometry

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug. It is mainly metabolized by phase 1 and 2 reactions in the liver, and thus it could be involved in many drug–drug interactions. Therefore, the study of APAP metabolism is important in toxicological and pharmacokinetic studies. The objective of this study was to develop a rapid and sensitive method for the determination of APAP and its six metabolites in rat plasma for the pharmacokinetic studies. APAP and its metabolites were separated through a Capcell Pak MGII C₁₈ column and quantitated with a 16 min run in a triple‐quadruple mass spectrometer. The mobile phases were composed of 0.1% formic acid in either 95% water or 95% acetonitrile and analysis was performed twice in positive and negative modes. Validations such as accuracy, precision, recovery, matrix effect and stability were found to be within acceptance criteria of validation guidelines, indicating that the assay was applicable to the determination of the plasma concentrations of drug and its six metabolites. In conclusion, we developed an LC‐MS/MS method for the quantitative analysis of APAP and its six metabolites in rat plasma, and this method appears to be useful for pharmacokinetic/toxicokinetic studies of APAP and its metabolites in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

Broad-spectrum drug screening of meconium by liquid chromatography with tandem mass spectrometry and time-of-flight mass spectrometry

Analysis of the major drugs of abuse in meconium has been established in clinical practice for detecting fetal exposure to illicit drugs, particularly for the ready availability of the sample and ease of collection from diapers, compared with neonatal hair and urine. Very little is known about the occurrence and detection possibilities of therapeutic and licit drugs in meconium. Meconium specimens (n = 209) were collected in delivery hospitals, from infants of mothers who were suspected to be drug abusers. A targeted analysis method by liquid chromatography–triple quadrupole mass spectrometry (LC-MS/MS) was developed for abused drugs: amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, morphine, codeine, 6-monoacetylmorphine, oxycodone, methadone, tramadol, buprenorphine, and norbuprenorphine. A separate LC-MS/MS method was developed for 11-nor-∆⁹-tetrahydrocannabinol-9-carboxylic acid. A screening method based on LC coupled to time-of-flight MS was applied to a broad spectrum of drugs. As a result, a total of 77 different compounds were found. The main drug findings in meconium were as follows: local anesthetics 82.5% (n = 172), nicotine or its metabolites 61.5% (n = 129), opioids 48.5% (n = 101), stimulants 21.0% (n = 44), hypnotics and sedatives 19.0% (n = 40), antidepressants 18.0% (n = 38), antipsychotics 5.5% (n = 11), and cannabis 3.0% (n = 5). By revealing drugs and metabolites beyond the ordinary scope, the present procedure helps the pediatrician in cases where maternal denial is strong but the infant seems to suffer from typical drug-withdrawal symptoms. Intrapartum drug administration cannot be differentiated from gestational drug use by meconium analysis, which affects the interpretation of oxycodone, tramadol, fentanyl, pethidine, and ephedrine findings.     

High-performance liquid chromatography/inductively coupled plasma mass spectrometry and tandem mass spectrometry for the detection of carbon-containing compounds

High-performance liquid chromatography (HPLC) combined with inductively coupled plasma mass spectrometry (ICPMS) has been studied as a means for the detection of carbon to provide a 'universal' method for detecting organic compounds in chromatographic eluents. Carbon is particularly difficult to ionise and the amount of carbon present in normal chromatographic systems leads to high backgrounds, making detection a challenge. Novel separation approaches were therefore employed, using either entirely aqueous eluents (at temperatures of 60 and 160 degrees C, dependent on the column used) to eliminate the organic modifier completely, or isotopically enriched solvents. For the aqueous eluents, detection limits for sulphanilamide were found to be 2.26 microg, corresponding to 1.13 micromol (0.47 micromol of carbon), injected on a conventional 4.6 mm i.d. column. The use of a narrow bore column with highly isotopically enriched 12C-methanol (99.95 atom%) as organic modifier for the mobile phase enabled the detection of 86 micromol for 13C-triple-labelled caffeine and 79 micromol for 13C-double-labelled phenacetin. The sensitive detection of 12C-compounds with 13C-enriched methanol as organic modifier proved impractical due to a lower level of isotopic enrichment (99 atom%) of this solvent, with the residual 12C-methanol resulting in significant interference.     

Open-access liquid chromatography/mass spectrometry in a drug discovery environment

The use of open-access mass spectrometry to monitor synthetic chemistry reactions, and also the integrity and purity of new chemical entities, has been a part of the medicinal chemist's tool-box for more than 5 years. Originally in our group at Wyeth Research there were two open-access methods available to the chemists, flow injection analysis (FIA) and liquid chromatography/mass spectrometry (LC/MS). The FIA method was approximately 3 min long, while the LC/MS method was approximately 20 min long (including an 8 min gradient). Within the first 2 years, the total number of open-access analyses increased by approximately 125%. It is interesting, however, that the number of LC/MS analyses increased by more than 285%. This is attributed to the fact that the chemists began using the LC/MS data to monitor reactions and also to check final product integrity and purity. In addition, the number of chemists performing parallel synthesis reactions has increased; thus, individual chemists can produce sample sets of up to 100 vials. This paper describes the implementation of new methodology, which accommodates the need for much faster run times and also the ability to acquire alternating positive and negative ion spectra within the same run. In addition, the instrument has been configured to e-mail the resulting processed data report to the submitting chemist. Several methods have been developed, including structure elucidation using in-source collision-induced dissociation (CID) and night-time analysis. The LC/MS methods for this system are described herein and are applicable to both industrial and academic synthetic chemistry optimization efforts.     

Quantitative determination of DNA adducts using liquid chromatography/electrospray ionization mass spectrometry and liquid chromatography/high-resolution inductively coupled plasma mass spectrometry

The quantitative determination of nucleotides from DNA modified by styrene oxide is described using a combination of inductively coupled plasma high-resolution mass spectrometry (ICP-HRMS) and electrospray ionization mass spectrometry (ESI-MS), both interfaced to reversed-phase high-performance liquid chromatography (HPLC). LC/ICP-MS (resolution > 1500 to discriminate against 15N16O+ and 14N16OH+) was employed to determine quantitatively the content of modified nucleotides in standard solutions based on the signal of phosphorus; phosphoric acid served as an internal standard. By means of the standard addition technique the sensitivity of the LC/ESI-MS approach was subsequently determined. Since a comparison of UV, ICP and ESI-MS data suggested that in ESI-MS the ionization efficiency of the adducts is identical within the error limits, quantitative determination of all adducts is possible. For LC/ESI-MS with single ion monitoring, the detection limit for styrene oxide adducts of nucleotides was determined to be 20 pg absolute or 14 modified in 10(8) unmodified nucleotides in a 5 micrograms DNA sample, which comes close to the best methods available for the detection of chemical modifications in DNA.     

Rapid screening for bisbibenzyls in bryophyte crude extracts using liquid chromatography/tandem mass spectrometry

A simple and rapid qualitative liquid chromatography-diode-array detection/tandem mass spectrometry (LC-DAD/MS/MS) method was developed and validated for screening bisbibenzyl compounds in bryophyte crude extracts at sub-ppm levels. After simple extraction with ethanol and analyte concentration with diethyl ether, the extracts were subjected to LC-DAD/MS/MS analysis. The overall instrument turnaround time was 50 min to obtain baseline separation of bisbibenzyl isomers in bryophytes. MS full scan, MS/MS precursor ion scan and MS/MS product ion scan modes were used for the screening. The bisbibenzyl standards studied gave limits of detection (LODs) at or below 10 ng/mL. The results also indicated that the method had acceptable precision to be used on a day-to-day basis for qualitative identification. The bisbibenzyl types, i.e. one biphenyl ether bond (A-type), two biphenyl ether bonds (B-type), one biphenyl ether and one biphenyl bond (C-type), or other biphenyl types can be differentiated by their ESI-MS/MS product profiles, and the number of alkoxyl substituents can also be identified. The linkage sites of biphenyl and biphenyl ether bonds cannot be identified for an unknown bisbibenzyl solely from its mass spectra. This system was used to support three screening assays of bryophytes including Marchantia polymorpha L., Ptagiochasm intermedium L. and Asterella angusta, which were collected from different places in China. From them, 7/12, 8/5 and 8/9 confirmed/unconfirmed bisbibenzyls were identified, respectively, based on their MS/MS data, UV spectra and the retention behavior. The screening method considerably reduced the time and the cost for the qualitative analyses, and the structure-fragmentation-UV relationships will facilitate the high-throughput screening (HTS) of bisbibenzyl compounds in bryophytes. It is also intended as a simple and convenient way for the determination of other structural families of natural products.     

Determination of 2-methoxyestradiol in human plasma, using liquid chromatography/tandem mass spectrometry

A liquid chromatography/tandem mass spectrometric (LC/MS/MS) assay was developed for the quantitative determination of 2-methoxyestradiol (2ME2) in human plasma. Sample pretreatment involved liquid-liquid extraction with ethyl acetate of 0.3-mL aliquots of plasma spiked with the internal standard, deuterated 2ME2 (2ME2-d5). Separation was achieved on a Zorbax Eclipse C18 column (2.1 x 50 mm, i.d., 5 microm) at room temperature using a gradient elution with methanol and water at a flow rate of 0.25 mL/min. Detection was performed using atmospheric pressure chemical ionization MS/MS by monitoring the ion transitions from m/z 303.1 --> 136.8 (2ME2) and m/z 308.1 --> 138.8 (2ME2-d5). Calibration curves were linear in the concentration range of 1-100 ng/mL. The accuracy and precision values, obtained from three different sets of quality control samples analyzed in quintuplicate on four separate occasions, ranged from 105-108% and from 3.62-5.68%, respectively. This assay was subsequently used for the determination of 2ME2 concentration in plasma of a patient with cancer after a single oral administration of 2ME2 at a dose of 2200 mg.     

Drug screening of pharmaceutical discovery compounds by micro-size exclusion chromatography/mass spectrometry.

Micro-size exclusion chromatography coupled with capillary liquid chromatography (capLC) and mass spectrometry (MS) provides a rapid and simple approach to the preliminary screening of active ligands toward a specific target macromolecule. In this study, the effectiveness of this technique is demonstrated by a number of small molecule ligands with known binding affinities towards the protein target. All ligands were incubated together with a target protein under native conditions. Separation was then achieved by microcentrifugation where the high molecular weight (MW) compounds were selectively passed through the size-exclusion material. The retained low MW compounds were then recovered and analyzed by capLC/MS. The absence of the ligand indicated strong affinity towards the target, while ligand detection indicated inactivity. This assay demonstrated the drugs that were acting as strong inhibitors of Co-PDF from those showing to be comparatively inactive. The relative binding rank order of the drugs towards Co-PDF was also determined. The results were validated by a corresponding set of control experiments in which the target molecules were excluded from the process. In principle, high-throughput micro-size exclusion chromatography, coupled with capLC/MS, offers a powerful technique as a preliminary screen in determining both the strong binding affinity and the relative affinity rank ordering of ligands towards a specific target macromolecule, and is complementary with other analytical drug screening techniques.     

Determination of Synacthen in human plasma using immunoaffinity purification and liquid chromatography/tandem mass spectrometry

Synacthen is a synthetic analogue to human adrenocorticotropin, which plays an important physiological role by stimulating production of cortisol. In sports, corticosteroids as well as releasing factors (corticotropins) are prohibited according to the regulations of the World Anti-Doping Agency, and the misuse of Synacthen has been reported several times. Hence, an assay enabling the detection of Synacthen in doping control samples has been developed using immunoaffinity chromatographic isolation of Synacthen from human plasma combined with a concentration of collected fractions using solid-phase extraction. Unambiguous determination of the target analyte was accomplished using microbore liquid chromatography/electrospray ionization tandem mass spectrometry. Diagnostic product ions such as m/z 223 were characterized using high-resolution/high-accuracy Orbitrap mass spectrometry and employed for triple quadrupole MS/MS analysis. The established assay requiring 2 mL of plasma allowed a lower limit of detection (LLOD) at 100 fmol/mL, a recovery of 97% and a precision at the LLOD < 20%. Authentic plasma samples obtained from a patient undergoing a standard short Synacthen test were used to prove the applicability of the developed procedure.     

Quantitative analysis of eight testosterone metabolites using column switching and liquid chromatography/tandem mass spectrometry

The rate at which testosterone is metabolized to different singly hydroxylated metabolites has been widely used as an in vitro marker for activity of different CYP450 enzymes. The interest in extra-hepatic metabolism, e.g. due to metabolism in the gut wall, has increased during the last decade. Measurement of extra-hepatic enzyme activity using testosterone as a substrate requires a highly sensitive analytical method. A new liquid chromatography/electrospray tandem mass spectrometry (LC/MS/MS) method, using column switching for online cleaning and desalting of samples, was developed and validated for analysis of 2alpha-, 2beta-, 6alpha-, 6beta-, 7alpha-, 16alpha-, and 16beta-hydroxytestosterone and androstenedione. The samples were injected on a SB-CN column and detection was performed using MS/MS. The limits of quantification ranged from 0.3 to 3.33 nM for the different metabolites. The validated method was used to quantify the enzyme activity in rat intestine mucosa. The formation rates of 16alpha-, 16beta-hydroxytestosterone and androstenedione were quantified, and 2beta-and 6beta-hydroxytestosterone were formed above the limits of detection.     

Quantitative determination of folic acid in multivitamin/multielement tablets using liquid chromatography/tandem mass spectrometry

Two different isotope-dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods for the quantitative determination of folic acid (FA) in multivitamin/multielement tablets are reported. These methods represent distinct improvements in terms of speed and specificity over most existing microbiological and chromatographic methods for the determination of FA in dietary supplements. The first method utilizes an aqueous/organic-based extraction solvent combined with positive-ion mode LC/MS/MS detection of protonated [M + H]+ FA molecules and the second method utilizes a pure aqueous-based extraction solvent combined with negative-ion mode LC/MS/MS detection of deprotonated [M - H]- FA molecules. The LC/MS/MS methods exhibit comparable linear dynamic ranges (> or =3 orders of magnitude), limits of detection (0.02 ng on-column) and limits of quantification (0.06 ng on-column) for FA. Two methods employing different extraction and different MS detection modes were developed to allow method cross-validation. Successful validation of each measurement procedure supports the use of either method for the certification of FA levels in dietary supplements. The accuracy and precision of each measurement procedure were evaluated by applying each method to the quantitative determination of FA in a NIST standard reference material (NIST SRM 3280 multivitamin/multielement tablets). The FA measurement accuracy for both methods was > or =95% (based on the manufacturer's assessment of the FA level in SRM 3280) with corresponding measurement precision values (% RSD) of approximately 1%.     

Quantitative determination of BMS-378806 in human plasma and urine by high-performance liquid chromatography/tandem mass spectrometry

BMS-378806 is a human immunodeficiency virus (HIV) entry inhibitor that is being developed for the oral treatment of HIV infection. Human plasma and urine LC/MS/ MS methods have been developed and validated for the quantitation of BMS-378806. For human plasma method, methyl t-butyl ether was used to extract BMS-378806 from plasma in a 96-well format, and the organic layers were dried down and then reconstituted for the injection, while a dilute-and-shoot approach was used for human urine method in a 96-well format. Chromatographic separation was achieved isocratically on a Phenomenex C18 (2) Luna column (2 x 50 mm2, 5 microm). The mobile phase contained 60:40 v/v of 0.1% formic acid in water and ACN. Detection was by positive ion electrospray MS/MS. The standard curves ranged from 1.25 to 1000 ng/mL for the plasma assay and from 10 to 5000 ng/mL for the urine assay. The curves were fitted to a 1/x2 weighted quadratic regression model for both methods. The validation results demonstrated that both methods had satisfactory precision and accuracy across the calibration ranges. The methods were applied to the analysis of human plasma and urine samples from a single ascending dose clinical study to assess the pharmacokinetics of the drug. The pharmacokinetic analysis results indicated the absorption and disposition of the drug was rapid. The systemic exposure of BMS-378806 was generally dose proportional among the doses from 100 to 1200 mg, but not dose proportional to 1600 mg. There were modest increases in the systemic exposure when the drug was given with food or given as a solution formulation. Renal excretion was not a substantial elimination pathway of the drug. BMS378806 was safe and well tolerated over a dose range of 100-1600 mg administered as a single oral dose.     

Simultaneous quantitative analysis of polyphenolic compounds in human plasma by liquid chromatography tandem mass spectrometry

A diet rich in polyphenolic compounds has recognized health benefits, and as such is routinely monitored as part of dietary intervention studies. A method for the simultaneous determination of 36 phenolic compounds, including phenolic acids and flavonoids, using liquid chromatography and tandem mass spectrometry is described here. The target analytes were quantified based on their specific mass spectral fragments using a selected reaction monitoring approach. A C18 column with embedded aromatic functionality ensured separation of all phenolic compounds studied which included several pairs of isomers. Sample preparation involved the use of β‐glucuronidase to release the phenolic compounds from their conjugated forms. The intra‐day and inter‐day precision and accuracy was less than 7% for all phenolic compounds studied. Recoveries, where plasma was spiked with three different concentrations of the analytes, ranged from 95–115%. The limits of detection and quantification were 0.23–3.89 and 1.15–7.79 nM, respectively. The method was successfully applied to real samples and the range reported for each phenolic compound, with the exception of hydroferulic acid, nordihydroguaiaretic acid, methylgallate, and m‐coumaric acid, was at least an order of magnitude higher than the limit of quantification for the method.