Determination of melamine and cyanuric acid in human urine by a liquid chromatography tandem mass spectrometry

Melamine was found to be the etiological factor for the urinary stones epidemic in infants and young children in China in 2008. Urine level of melamine and its analog cyanuric acid may be useful markers for the evaluation of toxic effects. Liquid chromatography tandem mass spectrometry methods for the individual determination of melamine and cyanuric acid in human urine are described. Using isotope labeled internal standards during liquid–liquid extraction, the method was fully validated by verifying specificity, linearity, LLOQ, intra- and inter-assay precision and accuracy, matrix effect, recovery and stability. Calibration curves with good linearity (r = 0.9999) over the concentration range from 10 to 5000 ng/ml, intra-assay precision <10% and inter-assay precision <15%, accuracy between 93.0 and 111.6% were obtained with multiple reaction monitoring mode for melamine and cyanuric acid in human urine. The methods were successfully applied to the analysis of urine samples collected from 86 infants and 110 adults.     

Simultaneous determination of six phenolic constituents of danshen in human serum using liquid chromatography/tandem mass spectrometry

The six phenolic constituents are water-soluble components extracted from the Chinese medical herb danshen, the dried roots of Salvia miltiorrhiza Bunge (Labiatae). An liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based method has been developed for the simultaneous quantification of six phenolic constituents of danshen (magnesium lithospermate B (MLB), rosmarinic acid (RA) and lithospermic acid (LA), caffeic acid (CAA), protocatechuic aldehyde (3,4-dihydroxybenzaldehyde, Pal), 3,4-dihydroxyphenyllactic acid (danshensu)) in human serum with chloramphenicol as internal standard. The serum samples were treated by special liquid-liquid extraction, and the analytes were determined using electrospray negative ionization mass spectrometry in the multiple reaction monitoring (MRM) mode, with sufficient sensitivity to allow analysis of human serum samples generated following administration of a clinically relevant dose. Good linearity over the range 8-2048 ng/mL for six phenolic constituents was observed. The intra- and inter-day precisions (CV) of analysis were <13%, and the accuracy ranged from 88 to 116%. This quantitation method was successfully applied to a pharmacokinetic study of i.v. drip infusion of Danshen injection fluid in human.     

Triazinic herbicide determination by gas chromatography-mass spectrometry in breast milk

A solid-phase extraction procedure using a graphitized carbon black cartridge for extraction and cleaning of a series of five triazines (atrazine, deethylatrazine, deisopropylatrazine, ametryne and prometryne) from breast milk samples was developed. Using a chemometric methodology, the optimisation of both the analysis time and the triazinic herbicide separation by gas chromatography-mass spectrometry (GC-MS) was then carried out with only 18 experiments. Detection and quantification limits for 1ml breast milk sample were, respectively, 0.3 and 1 ppb for each studied compound. The variation coefficients were less than 5% over the concentration range from 1 to 100 ppb. The accuracy was between 98.63 and 104.62% for each triazinic herbicide. The recovery was between 58.64 and 63.22% for the concentration range from 1 to 100 ppb for each triazinic herbicide. The assay was successfully applied to the analysis of several breast milk samples.     

Quantitative analysis of myo-inositol in urine, blood and nutritional supplements by high-performance liquid chromatography tandem mass spectrometry

Myo-inositol plays key physiological functions, necessitating development of methodology for quantification in biological matrices. Limitations of current mass spectrometry-based approaches include the need for a derivatisation step and/or sample clean-up. In addition, co-elution of glucose may cause ion suppression of myo-inositol signals, for example in blood or urine samples. We describe an HPLC-MS/MS method using a lead-form resin based column online to a triple quadrupole tandem mass spectrometer, which requires minimum sample preparation and no derivatisation. This method allows separation and selective detection of myo-inositol from other inositol stereoisomers. Importantly, inositol was also separated from hexose monosaccharides of the same molecular weight, including glucose, galactose, mannose and fructose. The inter- and intra-assay variability was determined for standard solutions and urine with inter-assay coefficient of variation (CV) of 1.1% and 3.5% respectively, while intra-assay CV was 2.3% and 3.6%. Urine and blood samples from normal individuals were analysed.     

Selective and sensitive determination of amisulpride in human plasma by liquid chromatography-tandem mass spectrometry with positive electrospray ionisation and multiple reaction monitoring

The development of a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with positive electrospray ionisation (ESI(+)) and multiple reaction monitoring (MRM) for the selective and sensitive bioanalytical determination of amisulpride, a substituted benzamide derivative, in human plasma is described. Plasma was cleaned up using a liquid-liquid extraction (diisopropylether:dichloromethane=1:1 (v/v)) procedure. The chemically related drug sulpiride was used as internal standard (ISTD) and a primary calibration function was established in the concentration range of 0.50-500.52 ng/ml for amisulpride in plasma by triple analysis of the corresponding calibration standards. A linear relationship between concentration and signal intensity (given as peak area ratio analyte/ISTD) was obtained (linear regression: r=0.9999). A lower limit of quantification (LLQ) of 0.50 ng/ml was used during measurement of study plasma samples. Satisfying results of within-day precision (CV=0.79 to 1.98%) and accuracy (mean relative deviation: -1.68 to 3.58%) and between-day precision (CV=1.34 to 4.62%) and accuracy (mean relative deviation: -1.73 to -3.77%) as well as of recovery (amisulpride: 81.74 to 84.83%; sulpiride: 58.65%) and selectivity investigations confirmed the high reliability of this established LC-MS/MS method. Sufficient stability of amisulpride in plasma was achieved during freeze-thaw-cycles, for storage periods of 24h at room temperature and 20 days at <-20 degrees C as well as in extracts (storage conditions: <-20 degrees C, 6 days and 7 degrees C, 6 days) with mean relative deviations between - 2.83 and 2.91%. An example of a pharmacokinetic profile determined after administration of an amisulpride 200mg dose in a pilot study is given in this paper. A peak plasma concentration (C(max)) of 522.58 ng/ml was achieved after 3.55 h (t(max)). Corresponding values of areas under the plasma concentration-time curve (AUC) of 3405.35 ngh/ml (AUC(0-infinity)) and 3306.54 ngh/ml (AUC(0-tlast)) were obtained. The terminal plasma elimination half-life (t(1/2)) was 10.36 h.     

Absolute quantification of the model biomarker prostate-specific antigen in serum by LC-Ms/MS using protein cleavage and isotope dilution mass spectrometry

Protein cleavage-isotope dilution mass spectrometry (PC-IDMS) can be used to quantify proteins, with an isotope-labeled analogue of the peptide fragment used as an internal standard. Here, we investigate use of a standard LC-MS/MS platform for quantifying a model biomarker directly from serum by this technique. We synthesized a peptide (IVGGWECEK) identical to the N-terminal tryptic fragment of PSA but with each glycine containing two 13C atoms and one 15N atom. PSA-free human serum was denatured with urea followed by the introduction of PSA standard and the stable isotope labeled internal standard peptide. The sample was then proteolyzed with trypsin and subjected to quantification using LC-MS/ MS on a triple quadrupole mass spectrometer. A linear least squares calibration curve made from five different concentrations of PSA added to serum and digested (each made in triplicate and randomly injected three times) had a mean slope of 0.973 (SE = 0.023), intercept of -0.003 (SE = 0.022), and R2 of 0.971. Recovery of calibrators ranged from 70 to 85% with a mean run-to-run CV of 13% and a mean within-run CV of 5.7%. PC-IDMS is a promising technique for quantifying proteins covering a broad range of applications from standardizing immunoassays to monitoring post-translational modifications to quantifying newly discovered biomarkers prior to the development and implementation of an immunoassay, just to name a few. Issues surrounding the application of PC-IDMS for the absolute quantification of proteins include selection of a proteolytic fragment for quantification that can be cleaved and isolated reproducibly over a broad dynamic range, stable isotope labeled synthetic peptide standards that give consistent results, and LC-MS/MS methods that provide adequate sensitivity and reproducibility without creating impractical analysis times. The results presented here show that absolute quantification can be performed on the model biomarker PSA introduced into denatured serum when analyzed by LC-MS/MS. However, concerns still exist regarding sensitivity compared to existing immunoassays as well as the reproducibility of PC-IDMS performed in different matrixes.     

Determination of serum uric acid using high-performance liquid chromatography (HPLC)/isotope dilution mass spectrometry (ID-MS) as a candidate reference method

Uric acid is an important diagnostic marker of catabolism of the purine nucleosides, and accurate measurements of serum uric acid are necessary for proper diagnosis of gout or renal disease appearance. A candidate reference method involving isotope dilution coupled with liquid chromatography/mass spectrometry (LC/MS) has been described. An isotopically labeled internal standard, [1,3-(15)N(2)] uric acid, was added to serum, followed by equilibration and protein removal clean up to prepare samples for liquid chromatography/mass spectrometry electrospray ionization (LC/MS-ESI) analyses. (M-H)(-) ions at m/z 167 and 169 for uric acid and its labeled internal standard were monitored for LC/MS. The accuracy of the measurement was evaluated by a comparison of results of this candidate reference method on lyophilized human serum reference materials for uric acid (Standard Reference Materials SRM909b) with the certified values determined by gas chromatography/mass spectrometry reference methods and by a recovery study for the added uric acid. The method performed well against the established reference method of ion-exchange followed by derivatization isotope dilution (ID) gas chromatography mass spectrometry (ID-GC/MS). The results of this method for uric acid agreed well with the certified values and were within 0.10%. The amounts of uric acid recovered and added were in good agreement for the three concentrations. This method was applied to determine uric acid in samples of frozen serum pools. Excellent precision was obtained with within-set CVs of 0.08-0.18% and between-set CVs of 0.02-0.07% for LC/MS analyses. Liquid chromatography/tandem mass spectrometry electrospray ionization (LC/MS/MS-ESI) analysis was also performed. The LC/MS and LC/MS/MS results were in very good agreement (within 0.14%). This LC/MS method, which demonstrates good accuracy and precision, and is in the speed of analysis without the need for a derivatization stage, qualifies as a candidate reference method. This method can be used as an alternative reference method to provide an accuracy base to which the routine methods can be compared.     

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.     

Validation of ELISA for the determination of anti-ricin immunoglobulin G concentration in mouse sera.

An enzyme-linked immunosorbent assay (ELISA) for the determination of anti-ricin immunoglobulin G (IgG) concentration in mouse sera was systematically validated. The results obtained throughout the validation process strongly demonstrated that the ELISA was reliable, reproducible, and suitable for its intended use. The assay had a high level of precision within and between runs, was specific for the anti-ricin IgG, and showed no interference with a number of different serum matrices. The assay exhibited excellent accuracy, linearity, and stability. The mean recovery of four test samples with different known concentrations was 100.9+/-11.3%, 102.7+/-10.8%, 99.0+/-7.2%, and 95.9+/-11.3%, respectively (n=10). The mean recovery of the observed anti-ricin IgG concentration of three quality control samples run on 73 plates to their nominal concentrations was 100.1+/-7.3%, 100.2+/-5.8%, and 103.7+/-8.1%; and the coefficient of variation (CV) was 7.3%, 5.8%, and 7.8%, respectively. The back-calculated anti-ricin IgG concentration, %CV, and relative error of seven standards from the calibration curves run in the entire validation study were analyzed (n=7 x 73). The results indicated that the four-parameter logistic (4PL) equation, y=(a-d)/(1+(x/c)b)+d, provided an accurate representation of a sigmoidal relationship between the measured response and the logarithm of observed concentration of anti-ricin IgG in mouse sera for this ELISA. The lower limit of quantification and upper limit of quantification of the calibration curve were 3.3 ng/ml and 82.8 ng/ml, respectively. The measurable range of the assay would cover all possible anti-ricin IgG concentrations in mouse sera stimulated with a ricin vaccine candidate, when the test sera are measured at a 1:800 starting dilution followed by four additional fourfold serial dilutions.     

Hemolysis of human erythrocytes induced by melamine-cyanurate complex

Melamine is a widely-used chemical in industries. In recent years, melamine has been found to be involved in outbreaks of renal injury in infants and animals. Pathological studies indicated that the melamine-induced acute renal failure was related to the concurrence of melamine and other triazine analogs such as cyanuric acid. In the present study, human erythrocytes were used as an in vitro model to explore the cytotoxicity of melamine and its complex with cyanuric acid. The results demonstrated that mixing melamine and cyanuric acid resulted in the formation of insoluble particles and that the insoluble melamine-cyanurate complex induced membrane damages of human erythrocytes. The membrane damages included hemolysis, K⁺ leakage, alterations in cell shape and membrane fragility, and inhibition of enzymatic activity. By contrast, either melamine or cyanuric acid alone had no effect on erythrocyte membranes. The results of this study may provide a fresh insight into the melamine toxicology.     

Generic solid phase extraction-liquid chromatography-tandem mass spectrometry method for fast determination of drugs in biological fluids

A generic method was developed for the fast determination of a wide range of drugs in serum or plasma. The methodology comprises generic solid-phase extraction, on-line coupled to gradient HPLC with tandem mass spectrometric detection (SPE-LC-MS/MS). The individual components of the SPE-LC-MS/MS system were optimized in an integrated approach to maximize the application range and minimize the method development time. The optimized generic SPE-LC-MS/MS protocol was evaluated for 11 drugs with different physicochemical properties. Good quantification for 10 out of 11 of the pharmaceuticals in serum or plasma could be readily achieved. The quantitative assays gave recoveries better than 95%, lower quantification limits of 0.2-2.0 ng/ml, acceptable precision and accuracy and good linearity over 2-4 orders of magnitude. Carry-over was determined to be in the range of 0.02-0.10%, without optimization.     

Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS

We describe a highly sensitive and specific method for the quantification of key regulatory oxysterols in biological samples. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After alkaline hydrolysis of human serum (5 microl) or rat liver microsomes (1 mg protein), oxysterols were extracted, derivatized into picolinyl esters, and analyzed by LC-MS/MS using the electrospray ionization mode. The detection limits of the picolinyl esters of 4beta-hydroxycholesterol, 7alpha-hydroxycholesterol, 22R-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and 24S,25-epoxycholesterol were 2-10 fg (5-25 amol) on-column (signal-to-noise ratio = 3). Reproducibilities and recoveries of these oxysterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.8% to 12.7% and 2.9% to 11.9%, respectively. The recovery experiments were performed using rat liver microsomes spiked with 0.05 ng to 12 ng of oxysterols, and recoveries of the oxysterols ranged from 86.7% to 107.3%, with a mean recovery of 100.6%. This method provides reproducible and reliable results for the quantification of oxysterols in small amounts of biological samples.     

Improved liquid chromatographic determination of serum cortisol with double internal standardization compared to radioimmunoassay and fluorometry, and evaluated by isotope dilution/mass spectrometry

A sensitive and specific high-performance liquid chromatographic (HPLC) method for the determination of cortisol in only 200 microliters of serum is described. Cortisol and two internal standards, 19-nortestosterone (IS1) and 6 alpha-methylprednisolone (IS2) are extracted with dichloromethane and analyzed on a C18 reversed-phase column eluted with a mobile phase of methanol:water at a flow rate of 0.75 ml/min. Ultraviolet absorption at 254 nm is used for detection and quantitation is performed by peak height ratio measurement. Using 200 microliters of serum, the lower limit of detection for cortisol is 10 ng/ml, the analytical recovery is 104 +/- 3.6% (n = 8), and the day-to-day precision was 1.69% at a level of 90 ng/ml (n = 16). Cortisol values obtained by this method were generally lower than those obtained by radioimmunoassay or by fluorometry. A serum pool was analyzed both by HPLC and by isotope dilution/mass spectrometry (ID/MS). A mean value of 90.1 ng/ml was obtained by HPLC (n = 16, CV = 1.7%), whereas ID/MS yielded a mean of 90.8 ng/ml (n = 28, CV = 0.4%). These results clearly demonstrate the high specificity and the accuracy of the HPLC procedure. The use of two internal standards not only compensates for losses during the sample manipulation but also prevents erroneous results in case of medication by either of these two products.     

Development and validation of a sensitive HPLC-ESI-MS/MS method for the direct determination of glucosamine in human plasma

A sensitive and specific HPLC-ESI-MS/MS method for the direct determination of glucosamine in human plasma has been developed and validated. Plasma samples were analyzed after a simple, one-step protein precipitation clean-up with trichloroacetic acid using a polymer-based amino high-performance liquid chromatography (HPLC) column and a water/acetonitrile mobile phase elution gradient, with d-[1-(13)C]glucosamine as the internal standard. Detection was performed by mass spectrometry, using an electrospray source and employing multiple reaction monitoring to separately monitor glucosamine and the internal standard. The limit of quantification of the method was 10ng/ml of glucosamine and the calibration curve showed a good linearity up to 1000ng/ml. The precision (R.S.D.) and the accuracy (bias) of the method at the limit of quantification were 13.8 and 4.0%, respectively, and the mean recovery of glucosamine at three concentration levels was 101.6+/-5.7%. The method was applied for the determination of glucosamine concentrations in human plasma samples collected from untreated healthy volunteers and, in a separate bioavailability study, to evaluate plasma glucosamine pharmacokinetics profiles after oral administration of crystalline glucosamine sulfate.     

Concerns in the development of an assay for determination of a highly conjugated adsorption-prone compound in human urine

Concerns in pre-analytical handling of urine samples are discussed using a new KDR kinase inhibitor, 3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one (compound A), as an example of a case where high light sensitivity and low analyte recovery (high affinity for container surface) were found. The absence of these problems in plasma samples may be a result of the plasma protein content. Low recovery of the analyte from urine can be remedied by either changing the container or by using additives, such as bovine serum albumin (BSA) or non-ionic surfactant Tween-20. In the case of compound A, changing containers (polypropylene versus glass vial) or addition of BSA did bring analyte recovery up to 80%. However, the addition of 0.2% Tween-20 into urine quality controls (QCs) gave more than 95% analyte recovery, indicating effective reduction of analyte loss to the surface of containers. The urine assay using mixed-mode SPE and LC-MS/MS was not affected significantly by introducing Tween-20 into the samples. The mean SPE extraction recovery was 68.4% and matrix suppression of ionization on MS was less than 8% at all analyte concentrations. The linear range of the calibration curve was 0.5-400 ng/mL on PE Sciex API 3000 LC-MS/MS system. The assay intraday accuracy and precision were 92.1-104.8% and <4.2% (%CV), respectively. Urine QC samples, containing 0.2% Tween-20, gave excellent recovery after three cycles of freeze and thaw. Since analyte loss to its urine container surface is not unique to compound A (M. Schwartz, W. Kline, B. Matuszewski, Anal. Chim. Acta 352 (1997) 299-307; A.L. Fisher, E. DePuy, T. Shih, R. Stearns, Y. Lee, K. Gottesdiener, S. Flattery, M. De Smet, B. Keymeulen, D.G. Musson, J. Pharm. Biomed. Anal. 26 (2001) 739-752), we suggest an evaluation of the potential problem in the early stages of urine assay development to ensure reliable quantitation of analytes. The addition of Tween-20 can serve as a useful analytical tool to other analytes with similar situations.     

Liquid chromatography-tandem mass spectrometry validated method for the estimation of indapamide in human whole blood

A highly precise and sensitive method for the estimation of indapamide in human whole blood using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) is described. The method developed is validated in human whole-blood matrix, with a sensitivity of 0.5 ng/ml as lower limit of quantification. The procedure for the extraction of indapamide and glimepiride as internal standard (IS) involves haemolysis and deprotienation of whole blood using ZnSO(4) followed by liquid-liquid extraction using ethyl acetate. The sample extracts after drying were reconstituted and analysed by LC-MS/MS, equipped with turbo ion spray (TIS) source, operating in the positive ion and selective reaction monitoring (SRM) acquisition mode to quantify indapamide in human whole blood. The mean recovery for indapamide was 82.40 and 93.23% for IS. The total run time was 2.5 min to monitor both indapamide and the IS. The response of the LC-MS/MS method for indapamide was linear over the range of 0.5-80.0 ng/ml with correlation coefficient, r>or=0.9991. The coefficient of variance (% CV) at 0.5 ng/ml was 4.02% and the accuracy was well within the accepted limit of +/-20% at 0.5 ng/ml and +/-15% at all other concentrations in the linear range. This method is fully validated for the accuracy, precision and stability studies and also applied to subject-sample analysis of bioequivalence study for 1.5mg sustained-release (SR) formulations.     

Determination of a cyclooxygenase II inhibitor in human plasma by capillary gas chromatography with mass spectrometric detection

Sensitive methods based on capillary gas chromatography (GC) with mass spectrometric (MS) detection in a selected-ion monitoring mode (SIM) for the determination of a cyclooxygenase II (COX-II) inhibitor (3-isopropoxy-4-(4-methanesulfonylphenyl)-5,5'-dimethyl-5H-furan-2-one, I) in human plasma, in two concentration ranges of 0.1-20 and 5-1000 ng/ml, are described. Following liquid-liquid extraction, the residue, after evaporation of the organic phase to dryness, was reconstituted in acetonitrile (20 l) and part of the extract (1 l) was analyzed by GC/MS/SIM. The drug (I) and internal standard (II) were separated on a 25 mx0.2 mm capillary column with HP Ultra 1 (100% dimethylpolysiloxane, 0.33 m) phase and analyzed by MS/SIM monitoring ions at m/z 237 and 282 for I and II, respectively. The standard curve was linear within the lower concentration range of 0.1-20 ng/ml and the lower limit of quantification (LLOQ) in plasma was 0.1 ng/ml. Intraday coefficients of variation (CV, n=5) were 8.9, 4.2, 5.7, 3.1, 1.9, 1.9, and 4.4% at 0.1, 0.2, 0.5, 1.0, 5.0, 10, and 20 ng/ml, respectively. The standard curve was also linear within the higher concentration range of 5-1000 ng/ml and the LLOQ in plasma was 5 ng/ml. Intraday coefficients of variation (CV, n=5) were all below 9% at all concentrations within the standard curve range. The accuracy for I in human plasma was 91-112% and the recovery of I and II was greater than 70% at all concentrations within both standard curve ranges. The details of the assay methodology are presented.     

Standardized LC-MS/MS based steroid hormone profile-analysis

In order to overcome many limitations of immunoassays, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has the potential to find its place in the clinical laboratory medicine for quantification of steroid hormones. A prerequisite for the application of a new analytical procedure in clinical diagnostics is standardization to minimize analytical intra- and interlaboratory variability and inaccuracy. We evaluate a newly standardized HPLC-MS/MS assay in kit-format, developed for routine determination of 16 steroid hormones in human serum samples. Fifteen metabolites can be measured quantitatively, which include aldosterone, androstenedione, androsterone, corticosterone, cortisol, cortisone, 11-deoxycortisol, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), 17β-estradiol (E2), estrone (E1), etiocholanolone, 17α-hydroxyprogesterone (17OHP), progesterone, and testosterone. 11-Deoxycorticosterone is the only compound rated as semi-quantitative in this kit. The sample preparation is performed by solid phase extraction (SPE) on a 96-well plate. The standardized assay has been validated for human serum in terms of lower and upper limit of quantification (LLOQ 0.01-32 ng/mL, ULOQ 5-8000 ng/mL), linear correlation coefficient of calibration (R(2)>0.9966), intra- and inter-day precision (intra-day 1.1-8.8%, inter-day 5.2-14.8% and 8.2-18.6% for 11-deoxycorticosterone), accuracy (intra-day 88.3-115.5% and 109.3-128.2% for 11-deoxycorticosterone, inter-day 91.4-117.2% and 102.3-137.1% for 11-deoxycorticosterone), analytical total error (3.6-17.8%), proficiency test accuracy (85.4-113.4%), recovery (68-99%), and metabolite stability (freeze/thaw stability 95.5-108.1%, short term stability 86.9-107.2%). Inter-assay comparison with a routine reference HPLC-MS/MS assay and seven immunoassays demonstrates the outstanding high performance of this HPLC-MS/MS based kit by improvements in accuracy for progesterone, androstenedione, and 17OHP. Finally, results of two metyrapone tests demonstrate the potential of the standardized HPLC-MS/MS assay for the analysis of a comprehensive steroid hormone profile in clinical diagnostics.     

Electrospray ionization LC-MS/MS validated method to quantify griseofulvin in human plasma and its application to bioequivalence study

A simple, sensitive and rapid liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and validated to quantify griseofulvin in human plasma using propranolol hydrochloride as internal standard (IS). Samples were prepared using solid phase extraction and analysed without drying and reconstitution. The analytes were chromatographed on Hypersil, hypurity C18 reverse phase column under isocratic conditions using 0.05% formic acid in water:acetonitrile (30:70, v/v) as the mobile phase. Total chromatographic run time was 3.0 min. Quantitation was done on a triple quadrupole mass analyzer API-3000, equipped with turbo ion spray interface and operating in multiple reaction monitoring (MRM) mode to detect parent-->product ion transition for analyte and IS. The method was validated for sensitivity, matrix effect, accuracy and precision, linearity, recovery and stability studies. Linearity in plasma was observed over the concentration range 20-3000 ng/mL for griseofulvin. Lower limit of quantification (LLOQ) achieved was 20 ng/mL with precision (CV) less than 10% using 5 microL injection volume. The absolute recovery of analyte (87.36%) and IS (98.91%) from spiked plasma samples was consistent and reproducible. Inter-batch and intra-batch coefficients of variation across four validation runs (LLOQ, LQC, MQC and HQC) was less than 7.5%. The accuracy determined at these levels was within +/-4.2% in terms of relative error. The method was applied to a pilot bioequivalence study of 500 mg griseofulvin tablet in six healthy human subjects under fed condition.     

Quantification of cyanuric acid residue in human urine using high performance liquid chromatography–tandem mass spectrometry

Concern has increased about the resulting health effects of exposure to melamine and its metabolic contaminant, cyanuric acid, after infants in China were fed baby formula milk products contaminated with these compounds. We have developed a selective and sensitive analytical method to quantify the amount of cyanuric acid in human urine. The sample preparation involved extracting free-form cyanuric acid in human urine using anion exchange solid phase extraction. Cyanuric acid was separated from its urinary matrix components on the polymeric strong anion exchange analytical column; the analysis was performed by high performance liquid chromatography–tandem mass spectrometry using negative mode electrospray ionization interface. Quantification was performed using isotope dilution calibration covering the concentration range of 1.00–200 ng/mL. The limit of detection was 0.60 ng/mL and the relative standard deviations were 2.8–10.5% across the calibration range. The relative recovery of cyanuric acid was 100–104%. Our method is suitable to detect urinary concentrations of cyanuric acid caused by either environmental exposures or emerging poisoning events.