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.     

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.     

Direct cocktail analysis of drug discovery compounds in pooled plasma samples using liquid chromatography-tandem mass spectrometry

Direct plasma injection technology coupled with a LC-MS/MS assay provides fast and straightforward method development and greatly reduces the time for the tedious sample preparation procedures. In this work, a simple and sensitive bioanalytical method based on direct plasma injection using a single column high-performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) was developed for direct cocktail analysis of double-pooled mouse plasma samples for the quantitative determination of small molecules. The overall goal was to improve the throughput of the rapid pharmacokinetic (PK) screening process for early drug discovery candidates. Each pooled plasma sample was diluted with working solution containing internal standard and then directly injected into a polymer-coated mixed-function column for sample clean-up, enrichment and chromatographic separation. The apparent on-column recovery of six drug candidates in mouse plasma samples was greater than 90%. The single HPLC column was linked to either an atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI) source as a part of MS/MS system. The total run cycle time using single column direct injection methods can be achieved within 4 min per sample. The analytical results obtained by the described direct injection methods were comparable with those obtained by semi-automated protein precipitation methods within +/- 15%. The advantages and challenges of using direct single column LC-MS/MS methods with two ionization sources in combination of sample pooling technique are discussed.     

Application of liquid chromatography-tandem mass spectrometry for the determination of opioidmimetics in the brain dialysates from rats treated with opioidmimetics intraperitoneally

We have determined three opioidmimetics (compounds I-III) in the rat brain dialysates after intraperitoneal (i.p.) administration of compounds I-III using a liquid chromatography/mass spectrometry with tandem mass spectrometry (LC-MS/MS). The dialysate samples with methanol were directly analyzed by online column-switching liquid chromatography. Using multiple reaction monitoring (MRM, product ions m/z 421 of m/z 657 for compound I, m/z 421 of m/z 643 for compound II, and m/z 407 of m/z 629 for compound III) on LC-MS/MS with electrospray ionization (ESI), opioidmimetics in rat brain dialysates were determined. Calibration curves of the method showed a good linearity in the range of 10-100 ng/ml for each compound. The limit of determination was estimated to be ca. 1 ng/ml for compounds II and III, and ca. 5 ng/ml for compound I, respectively. The precision of analysis showed coefficients of variation ranging from 4.7 to 10.4% at compound III concentration (10-100 ng/ml) in Ringer's solution. As a result, the procedure proved to be very suitable for routine analysis. The method was applied to the analysis of three opioidmimetics in the brain dialysate samples from rats treated with these compounds.     

Determination of unbound vismodegib (GDC-0449) concentration in human plasma using rapid equilibrium dialysis followed by solid phase extraction and high-performance liquid chromatography coupled to mass spectrometry

A rapid equilibrium dialysis (RED) assay followed by a solid phase extraction (SPE) high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the quantitative determination of unbound vismodegib in human plasma was developed and validated. The equilibrium dialysis was carried out using 0.3 mL plasma samples in the single-use plate RED system at 37°C for 6h. The dialysis samples (0.1 mL) were extracted using a Strata-X-C 33u Polymeric Strong Cation SPE plate and the resulting extracts were analyzed using reverse-phase chromatography and positive electrospray ionization (ESI) mass spectrometry. The standard curve, which ranged from 0.100 to 100 ng/mL for vismodegib, was fitted to a 1/x(2) weighted linear regression model. The lower limit of quantitation (LLOQ, 0.100 ng/mL) was sufficient to quantify unbound concentrations of vismodegib after dialysis. The intra-assay precision of the LC-MS/MS assay, based on the four analytical QC levels (LLOQ, low, medium and high), was within 7.7% CV and inter-assay precision was within 5.5% CV. The assay accuracy, expressed as %Bias, was within ±4.0% of the nominal concentration values. Extraction recovery of vismodegib was between 77.9 and 84.0%. The assay provides a means for accurate assessment of unbound vismodegib plasma concentrations in clinical studies.     

Increase of the LC-MS/MS sensitivity and detection limits using on-line sample preparation with large volume plasma injection

Large volume injection (LVI) has systematically been studied to improve LC-MS/MS sensitivity (signal-to-noise ratio, or S/N) and detection limits. The method of LVI was combined with on-line solid phase extraction (on-line SPE) and LC-MS/MS detection for analysis of compounds directly in plasma. It was demonstrated that LVI of plasma with on-line SPE-LC-MS/MS allows for improvement of sensitivity and detection limits without compromising chromatographic peak shape and resolution and inducing significant matrix and signal suppression effects. Furthermore, sensitivity and detection limits improve linearly with the injection volume up to 100 microL. Quantification of the model compounds in plasma demonstrated comparable calibration curve statistics, precision and accuracy for 5, 50 and 100 microL plasma injections.     

An ultra-high sensitive bioanalytical method for plasma melatonin by liquid chromatography-tandem mass spectrometry using water as calibration matrix

For the endogenous substances with an ultra-low level in biological fluids, such as melatonin, the blank biological matrix is obviously not "blank". This problem leads to a serious issue of the bioanalytical methods development and validation by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). This work developed and validated an ultra-high sensitive bioanalytical method for plasma melatonin by LC-MS/MS using water as calibration matrix. The lower limit of quantitation of the method was verified to be 1.0 pg/mL and the method exhibited a linear range of 1-5000 pg/mL. Potential matrix effects, accuracy and precision were fully monitored and validated by two complementary quality control approaches respectively using water and the pooled plasma as matrix. The intra-run and inter-run precisions were less than 11.5% and 12.2%, respectively, and the relative error was below ± 13.8% for all of 5 quality control levels. The method was successfully applied to investigate the daytime (8:00 AM-8:00 PM) baseline level of endogenous plasma melatonin, as well as the pharmacokinetic profiles of exogenous melatonin after oral administration in beagle dogs.     

Validation of a high throughput method for serum/plasma testosterone using liquid chromatography tandem mass spectrometry (LC-MS/MS)

Testosterone, the major androgenic hormone in humans, is commonly measured to aid in the diagnosis of clinical conditions related to its excess or deficiency. In addition, testosterone measurements are used to monitor testosterone replacement-, or antiandrogen therapy. Most commonly, automated direct immunoassays have been used to measure testosterone in human serum. Their advantage compared with other methodologies, lies in high- and rapid sample throughput with minimal human intervention. However, many automated testosterone immunoassays suffer from poor accuracy at the low concentration levels (<50ng/dL) seen in women and children, or in men undergoing anti-androgen therapy. Our objective was to develop a LC-MS/MS method which measures testosterone in human serum while fulfilling the following criteria: Rapid pre-analytical sample processing with minimal manual sample manipulation; Minimize sample volume requirements; Accurate, precise and unambiguous measurement; Functional sensitivity of 5-10ng/dL; Sample throughput of at least 30 samples per hour. Our validation criteria for precision, accuracy, and linearity was to have accuracy and linearity within mean limits of +/-10%; Intra and inter-assay precision of <15% throughout the reporting range. We also wanted to compare our results to a previously validated LC-MS/MS assay which utilized a manual liquid-liquid extraction and to an automated commercial immunoassay (Bayer ACS:180). We describe here a sensitive and rapid testosterone assay based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) utilizing on-line sample extraction and multiplexing.     

Determination of higenamine in human plasma and urine using liquid chromatography coupled to positive electrospray ionization tandem mass spectrometry

Higenamine is an active ingredient of Aconite root in Chinese herbal medicine and might be used as a new agent for a pharmaceutical stress test and was approved to undergo clinical pharmacokinetic study. Therefore, there exists a need to establish a sensitive and rapid method for the determination of higenamine in human plasma and urine. This paper described a sensitive and rapid method based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for the determination of higenamine in human plasma and urine. Solid-phase extraction (SPE) was used to isolate the compounds from biological matrices followed by injection of the extracts onto an Atlantis dC18 column with isocratic elution. The mobile phase was 0.05% formic acid in water-methanol (40:60, v/v). The mass spectrometry was carried out using positive electrospray ionization (ESI) and data acquisition was carried out in the multiple reaction monitoring (MRM) mode. The method was fully validated over the concentration range of 0.100-50.0 ng/mL and 1.00-500 ng/mL in plasma and urine, respectively. The lower limits of quantification (LLOQs) were 0.100 and 1.00 ng/mL in plasma and urine, respectively. Inter- and intra-batch precision was less than 15% and the accuracy was within 85-115% for both plasma and urine. Extraction recovery was 82.1% and 56.6% in plasma and urine, respectively. Selectivity, matrix effects and stability were also validated in human plasma and urine. The method was applied to the pharmacokinetic study of higenamine hydrochloride in Chinese healthy subjects.     

Determination of aldosterone in serum by liquid chromatography-tandem mass spectrometry

Measurement of serum aldosterone is clinically important in the diagnosis of hypertension. While isotope dilution gas chromatography-mass spectrometry (ID-GC-MS) provides reliable results, it requires derivatization and is lengthy and time-consuming. Detection by liquid chromatography-mass spectrometry (LC-MS) is a potentially superior method. The analysis utilizes 0.5mL of serum. The samples were extracted with dichloromethane-ether. The extract was evaporated to dryness and aldosterone was analyzed by LC-MS/MS operating in the negative mode ESI after separation on a reversed-phase column. Aldosterone was also measured by RIA. The calibration curves for analysis of serum aldosterone exhibited consistent linearity and reproducibility in the range of 60-3000pmol/L. Interassay CVs were 4.3-7.5% at aldosterone concentrations of 97-993pmol/L. The lower limit of quantitation (LOQ) was 30pmol/L (signal to noise ratio=10). The mean recovery of the analyte added to serum ranged from 95 to 102%. The regression equation by LC-MS/MS (x) and RIA (y) method was: y=1.33x+185 (r=0.95; n=124). Sensitivity and specificity of the LC-MS/MS method for serum aldosterone offer advantages over GC-MS by eliminating derivatization. The novel method is rapid, reliable and simple to perform with a routine LC-MS/MS spectrometer. The sensitivity is adequate for patient samples. Aldosterone concentrations reported by nonextraction RIA were consistently higher than those produced by LC-MS/MS.     

Detection of new exemestane metabolites by liquid chromatography interfaced to electrospray-tandem mass spectrometry

Exemestane is an irreversible aromatase inhibitor used for anticancer therapy. Unfortunately, this drug is also misused in sports to avoid some adverse effects caused by steroids administration. For this reason exemestane has been included in World Anti-Doping Agency prohibited list. Usually, doping control laboratories monitor prohibited substances through their metabolites, because parent compounds are readily metabolized. Thus metabolism studies of these substances are very important. Metabolism of exemestane in humans is not clearly reported and this drug is detected indirectly through analysis of its only known metabolite: 17β-hydroxyexemestane using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and gas chromatography coupled to mass spectrometry (GC-MS). This drug is extensively metabolized to several unknown oxidized metabolites. For this purpose LC-MS/MS has been used to propose new urinary exemestane metabolites, mainly oxidized in C6-exomethylene and simultaneously reduced in 17-keto group. Urine samples from four volunteers obtained after administration of a 25mg dose of exemestane were analyzed separately by LC-MS/MS. Urine samples of each volunteer were hydrolyzed followed by liquid-liquid extraction and injected into a LC-MS/MS system. Three unreported metabolites were detected in all urine samples by LC-MS/MS. The postulated structures of the detected metabolites were based on molecular formulae composition obtained through high accuracy mass determination by liquid chromatography coupled to hybrid quadrupole-time of flight mass spectrometry (LC-QTOF MS) (all mass errors below 2ppm), electrospray (ESI) product ion spectra and chromatographic behavior.     

Multi-component plasma quantitation of anti-hyperglycemic pharmaceutical compounds using liquid chromatography-tandem mass spectrometry

Type-2 diabetes is a disorder characterized by disrupted insulin production leading to high blood glucose levels. To control this disease, combination therapy is often used. Hypoglycemic agents such as metformin, glipizide, glyburide, repaglinide, rosiglitazone, nateglinide, and pioglitazone are widely prescribed to control blood sugar levels. These drugs provide the basis for the development of a quantitative multianalyte bioanalytical method. As an example, a highly sensitive and selective multi-drug method based on liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed. This rapid, automated method consists of protein precipitation of 20 microL of plasma coupled with gradient HPLC elution of compounds using 10 mM ammonium formate buffer and 0.1% formic acid in acetonitrile as the mobile phases. MS/MS detection was performed using turbo ion spray in the positive ion multiple reaction monitoring (SRM) mode. A lower limit of quantitation (LLQ) in a range of 1.0-5.0 ng/mL was achieved for all analytes. The linearity of the method was observed over a 500-fold dynamic range. Drug recoveries ranged from 86.2 to 94.2% for all analytes of interest. Selectivity, sample dilution, intra-day and inter-day accuracy and precision, and stability assessment were evaluated for all compounds.     

Rapid and sensitive liquid chromatography-tandem mass spectrometry: assay development, validation and application to a human pharmacokinetic study

Rasagiline is a highly potent, selective and irreversible second-generation monoamine oxidase inhibitor with selectivity for type B of the enzyme (MAO-B). The present studies aimed at developing and validating a rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for determination of rasagiline in human plasma and urine. LC-MS/MS analysis was carried out on a Finnigan LC-TSQ Quantum mass spectrometer using positive ion electrospray ionization (ESI(+)) and selected reaction monitoring (SRM). The assay for rasagiline was linear over the range of 0.01-40 ng/mL in plasma and 0.025-40 ng/mL in urine. It took 5.5 min to analyze a sample. The average recoveries in plasma and urine samples were both >85%. The RSD of precision and bias of accuracy were less than 15% and 10%, respectively, of their nominal values based on the intra- and inter-day analysis. The developed method was proved to be suitable for use in clinical pharmacokinetic study after single oral administration of 0.5, 1 and 2 mg rasagiline mesylate tablets in healthy Chinese volunteers.     

Doping control analysis of trenbolone and related compounds using liquid chromatography-tandem mass spectrometry

Trenbolone (17β-hydroxy-estra-4,9,11-trien-3-one) and its derivatives such as 17α-methyltrenbolone represent a class of highly potent anabolic-androgenic steroids, which are prohibited in sports according to the regulation of the World Anti-Doping Agency (WADA). Due to marginal gas chromatographic properties of these compounds but excellent proton affinities resulting from a large and conjugated π-electron system, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been the method of choice for the detection of these analytes in sports drug testing. Recent findings of trenbolone and methyltrenbolone in doping control urine samples of elite athletes demonstrated the importance of a sensitive and robust analytical method, which was based on an enzymatic hydrolysis of target compounds, liquid-liquid extraction and subsequent LC-MS/MS measurement. Diagnostic product ions obtained after collision-induced dissociation of protonated molecules were found at m/z 227, 211, 199 and 198, which enabled targeted screening using multiple reaction monitoring. Using 7 model compounds (trenbolone, epitrenbolone, methyltrenbolone, ethyltrenbolone, propyltrenbolone, 17-ketotrenbolone and altrenogest), the established method was validated for specificity, lower limits of detection (0.3-3 ng/mL), recovery (72-105%), intraday and interday precision (≤20%).     

Bioanalytical liquid chromatography tandem mass spectrometry methods on underivatized silica columns with aqueous/organic mobile phases

This review article summarizes the recent progress on bioanalytical LC-MS/MS methods using underivatized silica columns and aqueous/organic mobile phases. Various types of polar analytes were extracted by using protein precipitation (PP), liquid/liquid extraction (LLE) or solid-phase extraction (SPE) and were then analyzed using LC-MS/MS on the silica columns. Use of silica columns and aqueous/organic mobile phases could significantly enhance LC-MS/MS method sensitivity, due to the high organic content in the mobile phase. Thanks to the very low backpressure generated from the silica column with low aqueous/high organic mobile phases, LC-MS/MS methods at high flow rates are feasible, resulting in significant timesaving. Because organic solvents have weaker eluting strength than water, direct injection of the organic solvent extracts from the reversed-phase solid-phase extraction onto the silica column was possible. Gradient elution on the silica columns using aqueous/organic mobile phases was also demonstrated. Contrary to what is commonly perceived, the silica column demonstrated superior column stability. This technology can be a valuable supplement to the reversed-phase LC-MS/MS.     

Simultaneous determination of 1,5-dicaffeoylquinic acid and its active metabolites in human plasma by liquid chromatography-tandem mass spectrometry for pharmacokinetic studies

1,5-Dicaffeoylquinic acid (1,5-DCQA), a potent HIV-1 integrase inhibitor, is undergoing an evaluation as a promising novel HIV therapeutic agent. Here, we report a simple, rapid and robust LC-MS/MS method for simultaneous determination of 1,5-DCQA and its two active metabolites, 1-caffeoyl-5-feruoylquinic acid (1,5-CFQA) and 1,5-O-diferuoylquinic acid (1,5-DFQA) in human plasma. The quantitation of the target compounds was determined by selected reaction monitoring (SRM) mode using electrospray ionization (ESI). Good linearity was obtained in the 3-500 ng/ml range for each analyte and the analytical method was validated in terms of specificity, precision, accuracy, recovery, stability and matrix effect. These assays gave R.S.D.% values for precision always lower than 13.8% and R.E.% values for accuracy between -8.9 and 0.9%. In addition, the specificity, extraction recovery, stability and matrix effect were satisfactory too. Using the measured plasma concentrations of 1,5-DCQA and its active metabolites in five healthy volunteers, pharmacokinetic profiles of 1,5-DCQA and its active metabolites were evaluated, which supported the clinical pharmacokinetic studies successfully. Due to its high sensitivity, specificity and simplicity, the method could be used for pharmacokinetic studies of both 1,5-DCQA and its active metabolite, and for routine monitoring of their levels in human plasma.     

Quantitation of iptakalim in human plasma by high-performance liquid chromatography-tandem mass spectrometry

This paper describes a rapid and sensitive analytical method for the quantitation of iptakalim, a novel antihypertensive drug, in human plasma. The method is based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) using sildenafil as internal standard. Sample preparation involved liquid-liquid extraction with dichloromethane-diethyl ether (2:3, v/v) in a basic environment. Chromatography was carried out on an amino column with a mobile phase consisting of acetonitrile-water (55:45, v/v, water containing 0.5% formic acid). Detection employed electrospray ionization (ESI) tandem mass spectrometry in the multiple-reaction-monitoring (MRM) mode. The assay was linear in the concentration range of 0.5-100 ng/ml with a lower limit of quantitation (LLOQ) of 0.5 ng/ml. Intra- and inter-day precision (R.S.D.) were <4.5% and <12.0%, respectively and the accuracy (R.E.) was in the range +/-5%. The method was successfully applied to a single oral dose pharmacokinetic study in human volunteers.     

Liquid chromatography-tandem mass spectrometry assay for the quantitation of beta-dihydroartemisinin in rat plasma

Dihydroartemisinin (DHA) is a sesquiterpene used in the world as an antimalarial. To evaluate the pharmacokinetics of dihydroartemisinin in rats, a sensitive and specific liquid chromatography/tandem mass spectrometric (LC-MS/MS) method was developed and validated for the quantitation of dihydroartemisinin in rat plasma. For detection, a Sciex API 4000 LC-MS/MS with a TurboIonSpray ionization (ESI) inlet in the positive ion-multiple reaction monitoring (MRM) mode was used. The plasma samples were pre-treated by a simple liquid-liquid extraction with diethyl ether. The statistical evaluation for this method reveals excellent linearity, accuracy and precision for the range of concentrations 0.2-100.0 ng/mL. The method had a lower limit of quantification (LLOQ) of 0.2 ng/mL for beta-dihydroartemisinin in 100 microL of plasma. The method was successfully applied to the characterization of the pharmacokinetic profile of beta-dihydroartemisinin in rats after oral administration.     

Determination of azatadine in human plasma by liquid chromatography/tandem mass spectrometry

A sensitive method using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) was developed and validated for the analysis of antihistamine drug azatadine in human plasma. Loratadine was used as internal standard (IS). Analytes were extracted from human plasma by liquid/liquid extraction using ethyl acetate. The organic phase was reduced to dryness under a stream of nitrogen at 30 °C and the residue was reconstituted with the mobile phase. 5 μL of the resulting solution was injected onto the LC-MS/MS system. A 4.6 mm × 150 mm, I.D. 5 μm, Agilent TC-C(18) column was used to perform the chromatographic analysis. The mobile phase consisted of ammonium formate buffer 0.010 M (adjusted to pH 4.3 with 1M formic acid)/acetonitrile (20:80, v/v) The chromatographic run time was 5 min per injection and flow rate was 0.6 mL/min. The retention time was 2.4 and 4.4 min for azatadine and IS, respectively. The tandem mass spectrometric detection mode was achieved with electrospray ionization (ESI) iron source and the multiple reaction monitoring (MRM) (291.3 → 248.2m/z for azatadine, 383.3 → 337.3m/z for IS) was operated in positive ion modes. The low limit of quantitation (LLOQ) was 0.05 ng/mL. The intra-day and inter-day precision of the quality control (QC) samples was 8.93-11.57% relative standard deviation (RSD). The inter-day accuracy of the QC samples was 96.83-105.07% of the nominal values.