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Results for “multiple reaction monitoring (MRM)”

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At least 19 recordsLinked to original sources

PubMed2005

Rapid detection and identification of N-acetyl-L-cysteine thioethers using constant neutral loss and theoretical multiple reaction monitoring combined with enhanced product-ion scans on a linear ion trap mass spectrometer.

A sensitive and specific liquid chromatography-mass spectrometry (LC-MS) method based on the combination of constant neutral loss scans (CNL) with product ion scans was developed on a linear ion trap. The method is applicable for the detection and identification of analytes with identical chemical substructures (such as conjugates of xenobiotics formed in biological systems) which give common CNLs. A specific CNL was observed for thioethers of N-acetyl-L-cysteine (mercapturic acids, MA) by LC-MS/MS. MS and HPLC parameters were optimized with 16 MAs available as reference compounds. All of these provided a CNL of 129 Da in the negative-ion mode. To assess sensitivity, a multiple reaction monitoring (MRM) mode with 251 theoretical transitions using the CNL of 129 Da combined with a product ion scan (IDA thMRM) was compared with CNL combined with a product ion scan (IDA CNL). An information-dependent acquisition (IDA) uses a survey scan such as MRM (multiple reaction monitoring) to generate "informations" and starting a second acquisition experiment such as a product ion scan using these "informations." Th-MRM means calculated transitions and not transitions generated from an available standard in the tuning mode. The product ion spectra provide additional information on the chemical structure of the unknown analytes. All MA standards were spiked in low concentrations to rat urines and were detected with both methods with LODs ranging from 60 pmol/mL to 1.63 nmol/mL with IDA thMRM. The expected product ion spectra were observed in urine. Application of this screening method to biological samples indicated the presence of a number of MAs in urine of unexposed rats, and resulted in the identification of 1,4-dihydroxynonene mercapturic acid as one of these MAs by negative and positive product ion spectra. These results show that the developed methods have a high potential to serve as both a prescreen to detect unknown MAs and to identify these analytes in complex matrix.

Acetylcysteine↗

PubMed2004

Multiresidue confirmation of beta-agonists in bovine retina and liver using LC-ES/MS/MS.

Misuse of numerous beta-agonist drugs for their growth promoting effects in livestock production requires significant regulatory enforcement activities worldwide. The proof of illegal drug use needed for regulatory action usually requires the high degree of specificity derived from mass spectrometric analysis of suspect tissues and body fluids. In this paper, we describe a multiresidue screening method for confirmation of nine beta-agonist compounds in bovine liver and retina. A wide range of analyte structures was selected in order to demonstrate applicability to other chemically related beta-agonists for which standards are not currently available. The class-specific method, which is based on mixed mode cation exchange/reverse phase solid phase extraction, reverse phase gradient LC separation using a cyanopropyl-silica phase, and tandem mass spectrometry (MS/MS) in the multiple reaction monitoring (MRM) mode, yields high analyte recoveries at the target level of 1 ppb (ng/g). In addition, acquisition of multiple MRM transitions for each analyte permits simultaneous confirmation of beta-agonists at the level of 1 ppb in liver and retina by using intensity ratios between fragment ions and protonated molecules. Estimated values for the limit of quantification (LOQ) for individual beta-agonists were 0.08-0.3 ppb in liver and 0.02-0.5 in retina; the estimated limits of confirmation, using accepted criteria from international regulatory agencies, were 0.25-0.8 ppb in liver and 0.1-1 ppb in retina. This method should be useful in supporting regulatory enforcement programs that monitor beta-agonist misuse.

Adrenergic beta-Agonists↗

PubMed2001

Analysis of enzyme kinetics using electrospray ionization mass spectrometry and multiple reaction monitoring: fucosyltransferase V.

An accurate, rapid, and versatile method for the analysis of enzyme kinetics using electrospray ionization mass spectrometry (ESI-MS) has been developed and demonstrated using fucosyltransferase V. Reactions performed in primary or secondary amine-containing buffers were diluted in an ESI solvent and directly analyzed without purification of the reaction products. Decreased mass resolution was used to maximize instrument sensitivity, and multiple reaction monitoring (MRM), in the tandem mass spectrometric mode, was used to enhance selectivity of detection. The approach allowed simultaneous monitoring of multiple processes, including substrate consumption, product formation, and the intensity of an internal standard. MRM gave an apparent K(m) for GDP-L-fucose (GDP-Fuc) of 50.4 +/- 5.5 microM and a k(cat) of 1.46 +/- 0.044 s(-1). Under the same conditions, the conventional radioactivity-based assay using GDP-[U-(14)C]Fuc as substrate gave virtually identical results: K(m) = 54.3 +/- 4.6 microM and k(cat) = 1.49 +/- 0.039 s(-1). The close correlation of the data showed that ESI-MS coupled to MRM is a valid approach for the analysis of enzyme kinetics. Consequently, this method represents a valuable alternative to existing analytic methods because of the option of simultaneously monitoring multiple species, the high degree of specificity, and rapid analysis times and because it does not rely on the availability of radioactive or chromogenic substrates.

Amino Sugars↗

PubMed2004

Quantitative analysis of squalamine, a self-ionization-suppressing aminosterol sulfate, in human plasma by LC-MS/MS.

The special physico-chemical property of squalamine enables the formation of intra- or inter-molecular non-volatile strong salt, which is difficult to ionize in a mass spectrometer's interface. A sensitive, accurate, precise, and specific method for the quantitative determination of this self ion-suppressing compound in human plasma has been developed and validated using high performance liquid chromatography (HPLC) coupled with positive electrospray tandem mass spectrometry (MS/MS). Solid phase extraction (SPE) technique was utilized to extract human plasma samples using the Waters Oasis HLB cartridges. Deuterated squalamine was used as the internal standard (IS). Positive multiple reaction monitoring (MRM) mode was used to achieve both sensitivity and selectivity. A quadratic linearity range over 5-1000 ng/ml, R > 0.999 was achieved. Performance of the method has been validated and met all the specifications set forth in the US Food and Drug Administration's May 2001 "Bioanalytical Method Validation Guidance for Industry". Different sample reconstitution solutions were found to have dramatic impact on sensitivity of mass spectrometer used to squalamine. This is the first quantitation method using a positive and true multiple reaction monitoring mode detection for squalamine.

Cholestanols↗

PubMed2002

The investigation and the use of high flow column-switching LC/MS/MS as a high-throughput approach for direct plasma sample analysis of single and multiple components in pharmacokinetic studies.

Recently direct plasma injection LC/MS/MS technique has been increasingly used in pharmaceutical research and development due to the demand for higher throughput of sample analyses. In this work, two on-line extraction methods including high flow LC/MS/MS and high flow column switching LC/MS/MS were investigated. The evaluations were conducted and focused on their performances with respect to peak responses, separation efficiency, and signal to-noise ratio in a multiple-component LC/MS/MS assay. Two HPLC pumps were used-with one for high flow delivery and one for gradient elution. A CTC autosampler was used to inject plasma samples. High flow LC was achieved by the use of 4 ml/min flow rate on a 1 x 50 mm Waters Oasis column. A 2 x 100 mm YMC column was coupled via a column-switching valve. The extracted analytes were analyzed in multiple-reaction-monitoring (MRM) mode using a triple quadrupole MS/MS. As a rapid and simple procedure, vortex-mixing plasma and internal standard directly in sample vials completed sample preparation. The high flow column switching method (two-column system) provided sharper peak shape than the conventional high flow method. This effect increased analyte signal-to-noise ratio and sensitivity. Narrower peak width resulted in much better separation efficiency, which was required for multiple compound (N-in-1) analysis. A 2 mm I.D. column resulted in better peak shape and resolution than using a smaller I.D. column. The selected method achieved acceptable recoveries for most of the compounds tested, and it was successfully applied to a 10-in-1 pharmacokinetic (PK) study. The results showed that the dynamic range, lower limit of quantitation, assay accuracy and precision were acceptable for all compounds. Rapid sample preparation eliminated labor intensive and time consuming processes and improved productivity. This high throughput on-line extraction high flow column switching method has been proven particularly useful for multiple component analysis in PK studies.

Chromatography, Liquid↗

PubMed2024

Development of a Fit-For-Purpose Multi-Marker Panel for Early Diagnosis of Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) suffers from a lack of an effective diagnostic method, which hampers improvement in patient survival. Carbohydrate antigen 19-9 (CA19-9) is the only FDA-approved blood biomarker for PDAC, yet its clinical utility is limited due to suboptimal performance. Liquid chromatography-mass spectrometry (LC-MS) has emerged as a burgeoning technology in clinical proteomics for the discovery, verification, and validation of novel biomarkers. A plethora of protein biomarker candidates for PDAC have been identified using LC-MS, yet few has successfully transitioned into clinical practice. This translational standstill is owed partly to insufficient considerations of practical needs and perspectives of clinical implementation during biomarker development pipelines, such as demonstrating the analytical robustness of proposed biomarkers which is critical for transitioning from research-grade to clinical-grade assays. Moreover, the throughput and cost-effectiveness of proposed assays ought to be considered concomitantly from the early phases of the biomarker pipelines for enhancing widespread adoption in clinical settings. Here, we developed a fit-for-purpose multi-marker panel for PDAC diagnosis by consolidating analytically robust biomarkers as well as employing a relatively simple LC-MS protocol. In the discovery phase, we comprehensively surveyed putative PDAC biomarkers from both in-house data and prior studies. In the verification phase, we developed a multiple-reaction monitoring (MRM)-MS-based proteomic assay using surrogate peptides that passed stringent analytical validation tests. We adopted a high-throughput protocol including a short gradient (<10 min) and simple sample preparation (no depletion or enrichment steps). Additionally, we developed our assay using serum samples, which are usually the preferred biospecimen in clinical settings. We developed predictive models based on our final panel of 12 protein biomarkers combined with CA19-9, which showed improved diagnostic performance compared to using CA19-9 alone in discriminating PDAC from non-PDAC controls including healthy individuals and patients with benign pancreatic diseases. A large-scale clinical validation is underway to demonstrate the clinical validity of our novel panel.

Humans↗

PubMed2000

Analysis of sphingomyelin, glucosylceramide, ceramide, sphingosine, and sphingosine 1-phosphate by tandem mass spectrometry.

Free sphingoid bases such as sphingosine, sphinganine, and the respective phosphorylated bases, as well as the complex sphingolipids ceramides, glucosylceramide, and sphingomyelin, all dissociate to form structurally distinctive product ions. For sphingomyelin these ions are characteristic of their phosphorylcholine headgroup and are observed at m/z 184. The other sphingolipids dissociate to form carbocations characteristic of their sphingoid base. For common mammalian sphingoid bases such as d18:1 or d18:0 these product ions are detected at m/z 264 or 266, respectively. However, changes in the sphingoid base chain length, degree of unsaturation, or other modifications may correspondingly result in a shift in m/z of [figure: see text] this product ion. Additionally, the kinetics that govern the formation of these product ions is affected by the presence of a delta 4 double bond. Thus, internal standards for each type of sphingoid base are required for quantitative data. Structurally distinctive product ions, when used with either precursor ion or constant neutral loss scans allow highly specific and sensitive methods for sphingolipid analysis. They serve to greatly reduce background chemical noise, and enhance detection of sphingolipids at very low concentrations. This occurs by allowing only those ions that dissociate to yield a specific product ion or neutral loss to be passed to the detector. Additionally, these scans reveal the exact combinations of headgroup, sphingoid base, and fatty acid in a complex mixture by mass. The free sphingoid bases and Cer readily decompose in the ion source, whereas GlcCer and SM do not. Finally, each individual sphingolipid species fragmented optimally at a different collision energy, precluding the use of either precursor ion or neutral loss scans for quantitation. Multiple reaction monitoring (MRM) experiments directly address the issues regarding accurate quantitation of sphingolipids that precursor ion and neutral loss scans cannot. In these experiments both ionization and dissociation parameters are optimized for each individual species. By detecting only specific precursor and product ion pairs instead of scanning wide m/z ranges maximum sensitivity is attained. Furthermore, relative ion abundance data are not biased with regard to instrumental parameters. At this point simple loop injections can be used with the MRM scanning methods developed to observe changes in sphingolipid type and quantity in crude extracts on a class-by-class basis. This, however, is labor intensive requiring multiple injections and multiple runs for each class in order to obtain a complete picture of all sphingolipids present. As an alternative [figure: see text] to loop injections, HPLC-MS/MS methods are being developed. In these methods sphingolipids are eluted by class, thus, each individually optimized MRM method can be used at specific times in an LC run. This provides a highly sensitive and accurate quantitation as well as a complete picture of all sphingolipids in a single run (Fig. 6). Additionally, this methodology is amenable to automation and can be used for high-throughput screening of multiple samples.

Ceramides↗

PubMed2003

Selective, sensitive and rapid liquid chromatography-tandem mass spectrometry method for the determination of alfuzosin in human plasma.

A selective, sensitive and rapid liquid chromatography-tandem mass spectrometry method for the determination of alfuzosin in plasma was developed. A PE Sciex API 2,000 triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, using TurboIonSpray with positive ionisation was used. Using prazosin as an internal standard, liquid-liquid extraction was followed by C(18) reversed-phase liquid chromatography and tandem mass spectrometry. The mean recovery for alfuzosin was 82.9% with a lower limit of quantification set at 0.298 ng/ml, the calibration range being between 0.298 and 38.1 ng/ml. This assay method makes use of the increased sensitivity and selectivity of tandem mass spectrometric (MS-MS) detection to allow for a more rapid (extraction and chromatography) and selective method for the determination of alfuzosin in human plasma than has previously been described. The assay method was used to quantify alfuzosin in human plasma samples generated in a multiple-dose (5 mg bd.) study at steady state.

Adrenergic alpha-Antagonists↗

PubMed2006

Utility of cleavable isotope-coded affinity-tagged reagents for quantification of low-copy proteins induced by methylprednisolone using liquid chromatography/tandem mass spectrometry.

Gene expression changes underlie important biological and pharmacological responses. Although mRNA expression profiling is routine, quantification of low-abundance proteins, which typically represent key effectors of responses, remains challenging. A novel strategy was developed for sensitive and accurate quantification of low-abundance proteins in highly complex biological matrixes. First, the cysteine specificity of cleavable isotope-coded affinity tags (cICAT) was employed to reduce the complexity of the digested proteome of tissue homogenates and to improve the quantification of low-abundance proteins. Second, cICAT-treated tissue samples were analyzed on a capillary LC coupled to an ion trap MS to screen for the subset of cICAT-peptides, derived from target proteins of interest, that was successfully labeled and retrieved. Third, putatively identified peptides derived from target proteins were synthesized, cICAT-labeled, and used both to optimize multiple reactions monitoring (MRM) analysis and to confirm chromatographic retention time and fragmentation pattern. Finally, batch quantification of target peptides was performed using MRM on a LC/triple-quad MS/MS using (12)C- (control) and (13)C (experimental)-cICAT-labeled tissue mixtures. The utility of this method was demonstrated by elucidating the time-course of tyrosine aminotransferase induction in the liver of rats following treatment with the corticosteroid methylprednisolone (MPL). This approach significantly improved quantitative sensitivity, and the linear range was 10-fold greater than published previously. An additional advantage is that archived samples may be reinterrogated to investigate the regulation of additional targets that become of interest. Stored samples were sucessfully reinterrogated to monitor the induction of ornithine decarboxylase, which is also an MPL-induced protein. To our knowledge, this is the first report of an ICAT-based method that is capable of quantifying low-abundance proteins in highly complex samples, such as tissue homogenates. The approach enables simultaneous quantification of multiple effector proteins induced by biological or pharmacological stimuli, and the processed samples can be interrogated repeatedly as additional targets of interest arise.

Affinity Labels↗

PubMed1996

Identification of talinolol metabolites in urine of man, dog, rat and mouse after oral administration by high-performance liquid chromatography-thermospray tandem mass spectrometry.

A new specific HPLC-TSP-MS/MS assay for identification of beta-blocking drug talinolol and its metabolites in urinary samples of man, dog, rat and mouse after single oral administration has been developed. Centrifuged urines were directly injected into the HPLC-TSP-MS system. Based on thermospray MS/MS studies of 10 reference compounds, several selective CID-MS/MS reactions were found, which were typical of substructure elements of potential phase I metabolites. In this way the differentiation of various stereochemical positions of the hydroxyl group in the cyclohexyl ring moiety of metabolites was possible. Renal metabolic profiles for all investigated species were generated by HPLC-multiple reaction monitoring (MRM). The detected metabolites were characterized by TSP full scan and MS/MS analysis in relation to synthesized reference compounds. The major metabolic pathway in all species results in the hydroxylation of the cyclohexylring moiety of talinolol. In dog urine, a phase II metabolite, the O-glucuronide of talinolol (I) was found. In order to identify this structure, the use of electrospray ionization was also necessary.

Adrenergic beta-Antagonists↗

PubMed1999

Integrating qualitative and quantitative liquid chromatography/tandem mass spectrometric analysis to support drug discovery.

During the early phase of a drug discovery process, in order to facilitate the selection of drug candidates from a discovery research program, a liquid chromatography tandem mass spectrometry (LC/MS/MS) strategy has been developed to assess the preliminary pharmacokinetics and metabolism of new drug entities. For pharmacokinetic studies using multiple reaction monitoring (MRM), the parent drug concentration and its 'suspected' (predictable) metabolites were monitored in the biological samples simultaneously. For metabolic identification, the general methodologies most frequently used to search for metabolites include full scan, precursor-ion scan, product-ion scan and neutral-loss scan. However, when the metabolites do not produce intense signals for tandem mass spectrometry, a more sensitive and selective assay has to be developed, and MRM would be the method of choice. Likewise, an intelligent guess as to which MRM transitions ought to be used for the metabolites will be considered, based on the product ion mass spectrum of the parent drug.

Acetonitriles↗

PubMed2000

Collision-induced dissociation of corticosteroids in electrospray tandem mass spectrometry and development of a screening method by high performance liquid chromatography/tandem mass spectrometry.

A screening method based on liquid chromatography/electrospray tandem mass spectrometry was developed in order to control the illegal use of corticosteroids as growth promoters in cattle. The objective was the detection of low residue levels of corticosteroids or metabolites in biological matrices. Relative to other studies published on this subject, the present work focused on enhancing specificity and sensitivity. Firstly, fragmentation of corticosteroids by collision-induced dissociation was studied. In positive mode, the losses of H(2)O for each hydroxyl group fixed on the molecule, as well as the loss of HF or HCl for halogenated compounds, were observed. For higher collision energy, fragmentations in the B, C and D rings were induced. The negative mode was found to be more specific, inducing a cleavage of the C(20)-C(21) bond with concomitant loss of formaldehyde (CH(2)O). Secondly, three acquisition methods in the negative mode were studied and evaluated, recorded signals being the parent ion [M + acetate](-) and the two daughter ions, [M - H](-) and [M - H - CH(2)O](-). For dexamethasone, MS/MS instrumental detection limits of fragment ion and neutral loss scans, and of multiple reaction monitoring (MRM), were 250, 20 and 5 pg injected, respectively. The MRM method was then evaluated with the objective of use for the detection of corticosteroid residues in biological samples (urine, hair, muscle) and for a metabolism study.

Acetates↗

PubMed2000

Determination of antimigraine compounds rizatriptan, zolmitriptan, naratriptan and sumatriptan in human serum by liquid chromatography/electrospray tandem mass spectrometry.

Development of a rapid, sensitive and selective method for the determination of antimigraine drugs from human serum is essential for understanding the pharmacokinetics of these drugs when administered concurrently. Solid phase extraction (SPE) using Oasis HLB was used to extract the drugs (sumatriptan, naratriptan, zolmitriptan and rizatriptan) and the internal standard bufotenine from serum. A method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed and validated to simultaneously quantitate these antimigraine drugs from human serum. The precursor and major product ions of the analytes were monitored on a triple quadrupole mass spectrometer with positive ion electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. The base peak in all the analytes is formed by alpha cleavage associated with protonation of the secondary amine. Mechanisms for the formation of the collision-induced dissociation products of these antimigraine compounds are proposed. Linear calibration curves were generated from 1-100 ng/mL with all coefficients of determination greater than 0.99. The inter- and intraday precision (%RSD) were less than 9.3% and accuracy (%error) was less than 9.8% for all components. The limits of detection (LOD) for the method were 250 pg/mL for sumatriptan and 100 pg/mL for the remaining analytes based on a signal-to-noise ratio of 3.

Bufotenin↗

PubMed2000

Trace analysis of estrogenic chemicals in sewage effluent using liquid chromatography combined with tandem mass spectrometry.

A rapid, selective, sensitive, and reproducible method for the analysis of environmental estrogens, either natural or synthetic, present in samples from sewage treatment works (STW) has been developed. Isolation of these compounds from STWs was performed by applying a simple extraction procedure using an ENVI-CARB cartridge (a graphitized non porous carbon black) as the solid-phase extraction (SPE) system. Liquid chromatography coupled with atmospheric pressure chemical ionization (APCI) and tandem mass spectrometry was used for detection. For the multiple reaction monitoring (MRM) mode, the [M + H](+) ion of estrone and the [M + H-H(2)O](+) ions of 17beta-estradiol, estriol and 17alpha-ethynylestradiol were selected as the precursors for collisionally induced dissociation (CID). The average recoveries from sewage final effluent samples ranged from 84 to 93% for low levels, and from 89 to 95% for high levels. The precision of the method ranged from 11 to 8% for low level and from 9 to 7% for high level samples. The lower level of quantitation for these estrogens in STW samples was determined at 0.5 ng/L for 17beta-estradiol and 17alpha-ethynylestradiol, and 1 ng/L for estrone and estriol, based on 1-L aliquots of sewage treatment works water, using the optimum tuning parameters for each individual selected precursor ion/product ion transition. Compared to a previous gas chromatography/mass spectrometry (GC/MS) method for the analysis of ten STW samples, this method was shown to provide higher sensitivity and lower time consumption.

Calibration↗

PubMed2001

Amlodipine bioequivalence study: quantification by liquid chromatography coupled to tandem mass spectrometry.

OBJECTIVE: To assess the bioequivalence of two amlodipine tablet formulations (Amlodipine 5 mg tablet from Merck S.A. Indústrias Químicas, Brazil as test formulation and Norvasc 5 mg tablet from Laboratórios Pfizer Ltd., Brazil as reference formulation) in 24 healthy volunteers of both sexes. METHODS: The study was conducted using an open, randomized two-period crossover design with a 4-week washout interval. Plasma samples were obtained over a 144 h period. Plasma amlodipine concentrations were analyzed by combined liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS) with positive ion electrospray ionization using multiple reaction monitoring (MRM). From the amlodipine plasma concentration vs time curves, the following pharmacokinetic parameters were obtained: AUC(last), AUC(0-inf) and C(max). The statistical interval proposed was 80-125% according to the US Food and Drug Administration Agency. RESULTS: The limit of quantification was 0.1 ng/ml for plasma amlodipine analysis. The geometric mean and the 90% confidence interval (CI) test/reference ratios were 101.2 (92.9-110.2%) for AUC(last), 99.6 (91.5-108.4%) for AUC(0-inf) and 98.5 (89.0-109.1%) for C(max). CONCLUSION: Since the 90% CI for AUC(last), AUC(0-inf) and C(max) ratios were within in the 80-125% interval proposed by the US FDA, it was concluded that Amlodipine 5 mg tablet (test formulation) was bioequivalent to Norvasc 5 mg tablet, in terms of both rate and extent of absorption.

Adolescent↗

PubMed2002

Determination of lamivudine/stavudine/efavirenz in human serum using liquid chromatography/electrospray tandem mass spectrometry with ionization polarity switch.

A high-performance liquid chromatography/tandem mass spectrometry (LC-MS-MS) method with ionization polarity switch was developed and validated in human serum for the determination of a lamivudine (3TC)/stavudine (d4T)/efavirenz combination HIV therapy. Solid phase extraction (SPE) was used to extract these anti-HIV drugs and internal standard aprobarbital. A gradient mobile phase consisting of acetonitrile and 20 mM ammonium acetate buffer with pH adjusted to 4.5 using glacial acetic acid was utilized to separate these drugs on a hexylsilane column (150 x 2.0 mm i.d.). The total run time between injections was 18 min. The precursor and major product ions of these drugs were monitored on a triple quadrupole mass spectrometer in the multiple reactions monitoring (MRM) mode. Ionization polarity was switched in the middle of the LC run allowing these anti-HIV drugs with different physicochemical properties to be detected simultaneously. The effect of ion suppression from human serum was studied and no interference with the analysis was noted. The method was validated over the range of 1.1-540 ng/mL for 3TC, 12.5-6228 ng/mL for d4T and 1.0-519 ng/mL for efavirenz. The method was shown to be accurate, with intra-day and inter-day accuracy less than 14.0% and precise, with intra-day and inter-day precision less than 13.1%. The extraction recoveries of all analytes were higher than 90%.

Alkynes↗

PubMedDate not supplied

Evolution of an open-access quantitative bioanalytical mass spectrometry service in a drug discovery environment.

Increased demand for assays for compounds at the early stages of drug discovery within the pharmaceutical industry has led to the need for open-access mass spectrometry systems for performing quantitative analysis in a variety of biological matrices. The open-access mass spectrometers described here are LC/MS/MS systems operated in 'multiple reaction monitoring' (MRM) mode to obtain the sensitivity and specificity required to quantitate low levels of pharmaceutical compounds in an excess of biological matrix. Instigation of these open-access systems has resulted in mass spectrometers becoming the detectors of choice for non-expert users, drastically reducing analytical method development time and allowing drug discovery scientists to concentrate on their core expertise of pharmacokinetics and drug metabolism. Setting up an open-access facility that effectively allows a user with minimal mass spectral knowledge to exploit the MS/MS capability of triple quadrupole mass spectrometers presents a significantly different challenge from setting up qualitative single stage mass spectrometry systems. Evolution of quantitative open access mass spectrometry within a pharmaceutical drug metabolism and pharmacokinetics group, from its beginnings as a single generic system to a series of specialist fully integrated walk-up facilities, is described.

Automation↗

PubMed2006

Liquid chromatographic-electrospray tandem mass spectrometric determination of clarithromycin in human plasma.

A rapid, sensitive and specific LC-MS-MS method has been developed for the determination of clarithromycin (CLA) in human plasma using roxithromycin (ROX) as the internal standard. Samples were prepared via liquid-liquid extraction with methyl tert-butyl ether (MTBE) and chromatographed on a Supelco RP(18) (4.6 x 50 mm, 3 microm particle size) column with a mobile phase consisting of acetonitrile:methanol:60 mM (pH 3.5) ammonium acetate buffer (32.5:32.5:35) at a constant flow rate of 0.8 mL/min. The run time was 3 min with retention times of approximately 1.65 and 1.70 min for CLA and ROX, respectively. Detection was performed on a PE Sciex API 365 mass spectrometer equipped with a turboionspray ionization source in multiple reaction monitoring (MRM) mode. The MRM pairs were m/z 748.5 --> m/z 158.2 for CLA and m/z 837.7 --> m/z 679.3 for ROX, respectively, with dwell times of 200 ms for each transition. The validated calibration curve range was 5.00-5000 ng/mL, based on 0.100 mL plasma sample volume with signal-to-noise ratio (S/N) greater than 60 for CLA at the lower limit of quantification level (5.00 ng/mL). The correlation coefficients (r(2)) of the calibration curves were better than or equal to 0.996. The inter-day (n = 18) precision and accuracy of the quality control (QC) samples were less than 3.58% RSD (relative standard deviation) and -10.8% bias, respectively. The intra-day (n = 6) precision and accuracy of the quality control samples were less than 5.0 and 12.6%, respectively. There was no significant deviation from the nominal values after a 10-fold dilution of high concentration QC samples using blank matrix. The QC samples were stable when left on the bench for 24 h or after three freeze-thaw cycles. The processed samples were also stable in HPLC autosampler at 10C for over 72 h. No matrix ionization suppression was observed when extracted blank matrix or reconstitution solvent was injected onto the system with post-column infusion of clarithromycin and roxithromycin. No carryover was observed when an extracted blank plasma sample was injected immediately after a 5000 ng/mL ULOQ (the upper limit of quantification) standard. The mean recovery was 81.5 and 78.3%, respectively, for clarithromycin and internal standard.

Chromatography, High Pressure Liquid↗