Determination of S-adenosylmethionine and S-adenosylhomocysteine in plasma and cerebrospinal fluid by stable-isotope dilution tandem mass spectrometry

BACKGROUND: Available methods for the determination of nanomolar concentrations of S:-adenosylmethionine (SAM) and S:-adenosylhomocysteine (SAH) in plasma and cerebrospinal fluid (CSF) are time-consuming. We wished to develop a method for their rapid and simultaneous measurement. METHODS: We used tandem mass spectrometry (MS/MS) for the simultaneous determination of SAM and SAH, with stable-isotope-labeled internal standards. The (13)C(5)-SAH internal standard was enzymatically prepared using SAH-hydrolase and [(13)C(5)]adenosine. The method comprises a weak anion-exchange solid-phase extraction procedure serving as clean-up step for the deproteinized plasma and CSF samples. After clean-up, samples were injected on a C(18) HPLC column, which was connected directly to the tandem mass spectrometer, operating in MS/MS mode. RESULTS: In plasma samples, the intraassay CVs for SAM and SAH were 4.2% and 4.0%, respectively, and the interassay CVs were 7.6% and 5. 9%, respectively. In CSF, the intraassay CVs for SAM and SAH were 6. 8% and 6.9%, respectively, and the interassay CVs were 4.2% and 5.5%, respectively. Mean recovery of SAM and SAH for both matrices at two concentrations was 93%. Detection limits for SAM and SAH in samples were 7.5 and 2.5 nmol/L, respectively. Concentrations of SAM and SAH in plasma from healthy subjects were within the previously reported ranges. In 10 CSF samples, the mean concentrations (range) were 248 (137-385) nmol/L for SAM and 11.3 (8.9-14.1) nmol/L for SAH. CONCLUSIONS: SAM and SAH can be analyzed by MS/MS, taking optimal advantage of the speed and high sensitivity and specificity of this relatively new analytical technique.     

A rapid HPLC-mass spectrometry cyclosporin method suitable for current monitoring practices

OBJECTIVES: Cyclosporin is an immunosuppressant drug with a narrow therapeutic window. Trough and 2-h post-dose blood samples are currently used for therapeutic drug monitoring in solid organ transplant recipients. The aim of the current study was to develop a rapid HPLC-tandem mass spectrometry (HPLC-MS) method for the measurement of cyclosporin in whole blood that was not only suitable for the clinical setting but also considered a reference method. METHODS: Blood samples (50 muL) were prepared by protein precipitation followed by C(18) solid-phase extraction while using d(12) cyclosporin as the internal standard. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface in positive ionization mode. RESULTS: The assay was linear from 10 to 2000 microg/L (r(2)>0.996, n=9). Inter-day analytical recovery and imprecision using whole blood quality control samples at 10, 30, 400, 1500, and 2000 microg/L were 94.9--103.5% and <7.7%, respectively (n=5). The assay had a mean relative recovery of 101.6%. Ion suppression was<8.0% of the total signal (n=15). Extracted samples were stable for 6 h. Patient samples, measured by this method and compared with a validated HPLC-UV assay, revealed a strong correlation (r=0.998) and excellent agreement with a mean percentage bias of 2.1% (n=60). CONCLUSION: This high-throughput method provides accurate, precise, and specific measurement of cyclosporin in blood over a wide analytical range, thus making it suitable for current clinical monitoring strategies.     

Quantification of sirolimus by liquid chromatography-tandem mass spectrometry using on-line solid-phase extraction.

Quantification of the new immunosuppressant sirolimus (syn. rapamycin; Rapamune) in whole blood by chromatography is essential for its clinical use since no immunoassay is available although monitoring is mandatory. Here we report on a rapid and convenient liquid chromatography (LC-tandem mass spectrometry method and describe our practical experience with its routine use. Whole blood samples were hemolyzed and deproteinized using an equal volume (150 microl) of a mixture of methanol/zinc sulfate solution containing the internal standard desmethoxy-rapamycin. After centrifugation, the clear supernatants were submitted to an on-line solid-phase extraction procedure using the polymeric Waters Oasis HLB material, with elution of the extracts onto the analytical column in the back-flush mode by column switching. For analytical chromatography a RP-C18 column was used with 90/10 methanol/2 mM ammonium acetate as the mobile phase. A 1:10 split was used for the transfer to the mass spectrometer, a Micromass Quattro LC-tandem mass spectrometry system equipped with a Z-spray source and used in the positive electrospray ionization mode. The following transitions were recorded: sirolimus, 931>864 m/z, and desmethoxy-rapamycin (I.S.), 901>834 m/z. The analytical running time was 5 min, including on-line extraction. The method has a linear calibration curve (r>0.99; range 1.6-50 microg/l) and is rugged and precise with monthly CVs <7% at a sirolimus concentration of 13.1 microg/l in routine use; the instrumentation proved to be reliable and required minimal maintenance. Clin Chem     

Determination of trace elements and calcium in bone of the human iliac crest by atomic absorption spectrometry

A rapid and reliable analytical method for the determination of trace elements in human bone by atomic absorption spectrometry is reported. Calcium was determined to estimate the homogeneity of samples. Human bone from the iliac crest was obtained at autopsy of adult subjects. Before analysis samples were decomposed by microwave digestion and acid digestion in a Parr bomb. Zinc, rubidium, strontium, calcium and iron were determined by flame atomic absorption spectrometry (FAAS) and aluminium, copper and lead by electrothermal atomic absorption spectrometry (ETAAS) at optimum measurement conditions. The results for the two digestion procedures agreed for zinc, rubidium and calcium within +/-5%, for copper within +/-7% and for strontium, iron, aluminium and lead within +/-10%. The repeatability of measurement (R.S.D.) for determination of calcium and trace elements after microwave digestion and acid digestion in a Parr bomb was tested in one representative autopsy bone sample by six parallel determinations. It was found to be better than +/-5% either for microwave digested samples or samples digested in a Parr bomb, for all elements determined by FAAS and ETAAS techniques. The accuracy of the applied digestion procedures was checked by analysis of trace elements in NIST SRM 1486 Bone Meal reference material. Good agreement of the results with certified values was obtained for both digestion procedures. The microwave procedure developed for digestion of small amounts of sample was applied in trace elements analysis of bone biopsy samples from dialysis patients.     

A rapid liquid chromatography-tandem mass spectrometry analysis of whole blood sirolimus using turbulent flow technology for online extraction

BACKGROUND: Sirolimus is an immunosuppressant used in solid organ transplantation. Due to variable individual pharmacokinetics and narrow therapeutic ranges, therapeutic drug monitoring (TDM) is critical to the success of post-transplantation patient care. We developed a rapid method quantifying whole blood sirolimus using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with an automated online extraction technology. METHODS: Whole blood (100 microL) was mixed with a precipitation solution containing internal standard (32-desmethoxyrapamycin) and centrifuged at 15,634 x g for 10 min. The supernatant (50 microL) was injected onto a turbulent flow preparatory column and then a C18 analytical column. The mass spectrometer was set for positive electrospray to monitor the ammonium adducts. RESULTS: Analytical time was 4 min/injection. Inter- and intra-assay variation coefficients across three concentration levels ranged from 2.3% to 7.4%. The method was linear from 1.0 to 100.0 ng/mL with an accuracy of 93.3%-100.0%. No carryover was detected from samples at 313.6 ng/mL. There was no obvious ion suppression from patient samples or interference from other commonly used immunosuppressants. Good correlation with an in-house commercial LC-MS was observed. CONCLUSIONS: The LC-MS/MS method coupled with turbulent flow technology is rapid and efficient in TDM of whole blood sirolimus.     

Determination of amphetamine, methamphetamine, MDA and MDMA in human hair by GC-EI-MS after derivatization with perfluorooctanoyl chloride

The aim of this study was to develop a quantitative gas chromatography mass spectrometry (GC-MS) method to determine the classical amphetamines and their methylenedioxylated derivatives in human hair. The procedure involved liquid-liquid extraction of hydrolysed hair spiked with deuterated internal standards and direct derivatization with perfluorooctanoyl chloride. After evaporation of the organic phase and dissolution in butylacetate, the derivatized compounds were injected into a GC-MS. Method validation results showed a linear range from 0.25 to 25 ng/mg for the target compounds: amphetamine (AM), methamphetamine (MA), methylenedioxyamphetamine (MDA) and methylenedioxymethamphetamine (MDMA or ecstasy). An intra-day precision of 3-6% RSD and an inter-day precision of 3-17% RSD were observed. Trueness was between 96 % and 106% for the target compounds. The limit of detection ranged from 0.07 to 0.14 ng/mg and of quantification from 0.24 to 0.46 ng/mg, depending on compound. The method was applied on 40 authentic hair samples (segmented or pooled hair), of which 15 cases involved amphetamine and/or ecstasy. The hair concentrations ranged from LOD to 3.2 ng/mg of AM in 7 cases, to 0.4 ng/mg of MDA in 3 cases and to 5.9 ng/mg of MDMA in 13 cases. MA was only detected once at trace level. The method, including the derivatization procedure, is simple and robust with a sensitivity that is satisfactory for measurement of amphetamines and ecstasy in hair from abusers.     

Improvement of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of difficult-to-identify bacteria and its impact in the workflow of a clinical microbiology laboratory

This study evaluates matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) capability for the identification of difficult-to-identify microorganisms. A total of 150 bacterial isolates inconclusively identified with conventional phenotypic tests were further assessed by 16S rRNA sequencing and by MALDI-TOF MS following 2 methods: a) a simplified formic acid-based, on-plate extraction and b) performing a tube-based extraction step. Using the simplified method, 29 isolates could not be identified. For the remaining 121 isolates (80.7%), we obtained a reliable identification by MALDI-TOF: in 103 isolates, the identification by 16S rRNA sequencing and MALDI TOF coincided at the species level (68.7% from the total 150 analyzed isolates and 85.1% from the samples with MALDI-TOF result), and in 18 isolates, the identification by both methods coincided at the genus level (12% from the total and 14.9% from the samples with MALDI-TOF results). No discordant results were observed. The performance of the tube-based extraction step allowed the identification at the species level of 6 of the 29 unidentified isolates by the simplified method. In summary, MALDI-TOF can be used for the rapid identification of many bacterial isolates inconclusively identified by conventional methods.     

Drug testing in blood: validated negative-ion chemical ionization gas chromatographic-mass spectrometric assay for determination of amphetamine and methamphetamine enantiomers and its application to toxicology cases

BACKGROUND: Enantioselective analysis of amphetamine (AM) or methamphetamine (MA) in urine is already a well-established tool for differentiation of illicit from therapeutic ingestion of AM or MA derivatives. However, because of the increasing importance of plasma or serum in analytical toxicology, a method for enantioselective analysis of AM and MA in these matrices is needed. METHODS: AM and/or MA were extracted from 0.2 mL of blood plasma or serum by mixed-mode solid-phase extraction. After derivatization with S-(-)-heptafluorobutyrylprolyl chloride, the resulting diastereomers were separated by gas chromatography on a HP-5MS column during a 15-min program and detected by mass spectrometry in the negative-ion chemical ionization mode (NICI-GC-MS). The method was fully validated and applied to >50 samples from authentic toxicology cases. RESULTS: The derivatized AM and MA enantiomers were well separated and sensitively detected. The method was linear from 5 to 250 micro g/L per enantiomer with analytical recoveries, accuracy, and within- and between-run precision well within required limits. Extraction yields were 88.9-98.6%. Implications of concentrations and enantiomeric composition of AM and MA in the authentic samples were considered. CONCLUSIONS: This sensitive, reliable, rapid NICI-GC-MS assay is suitable for enantioselective determination of AM and MA in blood plasma or serum samples.     

Determination of amphetamine,methamphetamine, MDA and MDMA in human hair by GC‐EI‐MS after derivatization with perfluorooctanoyl chloride

The aim of this study was to develop a quantitative gas chromatography mass spectrometry (GC‐MS) method to determine the classical amphetamines and their methylenedioxylated derivatives in human hair. The procedure involved liquid–liquid extraction of hydrolysed hair spiked with deuterated internal standards and direct derivatization with perfluorooctanoyl chloride. After evaporation of the organic phase and dissolution in butylacetate, the derivatized compounds were injected into a GC‐MS. Method validation results showed a linear range from 0.25 to 25?ng/mg for the target compounds: amphetamine (AM), methamphetamine (MA), methylenedioxyamphetamine (MDA) and methylenedioxymethamphetamine (MDMA or ecstasy). An intra‐day precision of 3–6?% RSD and an inter‐day precision of 3–17?% RSD were observed. Trueness was between 96?% and 106?% for the target compounds. The limit of detection ranged from 0.07 to 0.14?ng/mg and of quantification from 0.24 to 0.46?ng/mg, depending on compound. The method was applied on 40 authentic hair samples (segmented or pooled hair), of which 15 cases involved amphetamine and/or ecstasy. The hair concentrations ranged from LOD to 3.2?ng/mg of AM in 7 cases, to 0.4?ng/mg of MDA in 3 cases and to 5.9?ng/mg of MDMA in 13 cases. MA was only detected once at trace level. The method, including the derivatization procedure, is simple and robust with a sensitivity that is satisfactory for measurement of amphetamines and ecstasy in hair from abusers.     

Analysis of pyrimidine synthesis "de novo" intermediates in urine and dried urine filter- paper strips with HPLC-electrospray tandem mass spectrometry

BACKGROUND: The concentrations of the pyrimidine "de novo" metabolites and their degradation products in urine are useful indicators for the diagnosis of an inborn error of the pyrimidine de novo pathway or a urea-cycle defect. Until now, no procedure was available that allowed the analysis of all of these metabolites in a single analytical run. We describe a rapid, specific method to measure these metabolites by HPLC-tandem mass spectrometry. METHODS: Urine or urine-soaked filter-paper strips were used to measure N-carbamyl-aspartate, dihydroorotate, orotate, orotidine, uridine, and uracil. Reversed-phase HPLC was combined with electrospray ionization tandem mass spectrometry, and detection was performed by multiple-reaction monitoring. Stable-isotope-labeled reference compounds were used as internal standards. RESULTS: All pyrimidine de novo metabolites and their degradation products were measured within a single analytical run of 14 min with lower limits of detection of 0.4-3 micromol/L. The intra- and interassay variation for urine with added compounds was 1.2-5% for urines and 2-9% for filter-paper extracts of the urines. Recoveries of the added metabolites were 97-106% for urine samples and 97-115% for filter-paper extracts of the urines. Analysis of urine samples from patients with a urea-cycle defect or pyrimidine degradation defect showed an aberrant metabolic profile when compared with controls. CONCLUSION: HPLC with electrospray ionization tandem mass spectrometry allows rapid testing for disorders affecting the pyrimidine de novo pathway. The use of filter-paper strips could facilitate collection, transport, and storage of urine samples.     

Validation of a rapid and sensitive liquid chromatography-tandem mass spectrometry method for free and total mycophenolic acid

BACKGROUND: Because mycophenolic acid (MPA) is highly protein bound and because the free fraction is the pharmacologically active portion, a rapid, reliable, and sensitive procedure is required to study the relationship between free MPA and treatment efficacy/toxicity. Liquid chromatography-tandem mass spectrometry is ideally suited for such a method. METHODS: Free MPA was isolated from plasma by ultrafiltration. An online extraction cartridge with a column-switching technique, analytical liquid chromatography over an Aqua Perfect C(18) column, and electrospray tandem mass spectrometry was used to quantify free and total MPA. To investigate ion suppression, a continuous infusion of MPA was introduced into the effluent from the HPLC column, and different ultrafiltrates and extracted plasma samples were injected on the column. RESULTS: A chromatographic run time of 4 min separated MPA from metabolites and internal standard, thereby avoiding interference from in-source fragmentation. Ion suppression occurred well before elution of MPA and internal standard. The lower limit of quantification for free MPA was 0.5 microg/L, and the method was linear to 1000 microg/L. Interassay imprecision (CV) was <10% for free MPA (0.5-333 microg/L). Agreement was good for free MPA (n = 52) and total MPA (n = 106) between the proposed method and a validated HPLC method with ultraviolet detection. The Passing-Bablok regression line was: y = 0.95x + 0.27 microg/L for free MPA and y = 0.98x + 0.03 mg/L for total MPA. CONCLUSIONS: The presented method allows the accurate, precise, and rapid determination of free and total MPA in plasma over a wide analytical range covering the concentrations relevant to pharmacokinetic studies and routine monitoring of this drug.     

Determination of coumarin-type anticoagulants in human plasma by HPLC-electrospray ionization tandem mass spectrometry with an ion trap detector.

BACKGROUND: Coumarin-type anticoagulants are used for the long-term treatment and prevention of thromboembolic disorders. The identification of these drugs is crucial in patients with an increased prothrombin time of unknown origin. The aim of this study was to develop a sensitive and specific method for the simultaneous determination of phenprocoumon, acenocoumarol, and warfarin in human plasma by HPLC-electrospray ionization tandem mass spectrometry. METHODS: After addition of the internal standard, p-chlorowarfarin, plasma samples were extracted using Oasis MCX solid-phase extraction cartridges. The compounds were separated on a Symmetry C18 column (Waters) with a mobile phase of acetonitrile-1 g/L formic acid (75:25 by volume) at a flow rate of 0.5 mL/min. RESULTS: Extraction and separation of the three drugs and the internal standard were accomplished in 9 min. The overall extraction efficiency was >89% for all three compounds. The limits of detection were 1 microg/L for phenprocoumon and warfarin and 10 microg/L for acenocoumarol. Regression analysis of the calibration data revealed good correlation (r(2) >or=0.995) for all compounds. Within-run accuracies for quality-control samples were +/- 1% to 7% of the target concentration, with CVs <9%. CONCLUSIONS: The proposed method enables the unambiguous identification and quantification of phenprocoumon, warfarin, and acenocoumarol in both clinical and forensic specimens. This method combines a new, rapid solid-phase extraction procedure with an extremely fast chromatographic analysis, which is especially advantageous for clinical laboratories.     

Free and bound enantiomers of methadone and its metabolite, EDDP in methadone maintenance treatment: relationship to dosage?

OBJECTIVES: To determine the effects of metabolism and protein binding on the relationship between administered dose, blood levels of R methadone and biological response by measuring the free and protein-bound forms of the R and S enantiomers of methadone and EDDP, its metabolite. DESIGN AND METHODS: To measure free and total drug, trough levels were collected from 45 methadone clients. To measure free methadone, samples were filtered using ultrafiltration with a MW weight cut-off of 10,000 and extracted using liquid-liquid extraction. The solvent was evaporated and samples reconstituted in mobile phase for analysis by LC/MS/MS. Total analyte was determined by extracting unfiltered samples. Enantiomeric separation of methadone and EDDP was by chiral chromatography. RESULTS: The presence of unmetabolized methadone suggested that none of the patients were very fast metabolizers. R and S forms were metabolized at the same rate at all administered doses. Free R methadone levels correlated both with methadone dose and with the total amount of R methadone. The free fraction of R methadone (%free R) was higher at lower doses than at high doses, varied from 5 to 25% and was inversely proportional to the total dose of administered drug in a relationship that was logarithmic and non-linear. CONCLUSIONS: By measuring the free, biologically active form of the drug, we were unable to account for the large variations in dose required between different patients to prevent the onset of withdrawal symptoms. The reason for the large range in dosage may be multifactorial.     

Determination of (S)- and (R)-2-oxo-3-methylvaleric acid in plasma of patients with maple syrup urine disease.

An enzymatic method for the separate measurement of both chiral 2-oxo-3-methylvaleric acid (OMV) compounds, (S)- and (R)-OMV, by NADH-dependent enantioselective amination using leucine dehydrogenase in the presence of a NADH regenerating system is described. This method allows the quantitative determination of all branched-chain 2-oxo acids, simultaneously. In plasma samples from classical maple syrup urine disease patients under therapy the average (R)-OMV/(S)-OMV ratio was 0.35 and great differences in the transamination equilibria of the diastereomeric branched-chain amino acids L-isoleucine and L-alloisoleucine were demonstrated.     

Rapid analysis of parathion in biological samples using headspace solid-phase micro-extraction (HS-SPME) and gas chromatography/mass spectrometry (GC/MS).

A simple and rapid method for the analysis of parathion in biological samples is presented. The method consists of the extraction of parathion from blood samples by headspace solid-phase micro-extraction (SPME), followed by capillary gas chromatography and mass spectrometry detection. The recoveries in the blood samples after addition of ammonium sulphate and sulphuric acid were between 85% and 89% compared to samples prepared in water. Linearity was established over a concentration range of 0.1-5 microg/g blood with acceptable coefficients of correlation and limits of detection reached 0.02-0.05 microg/g. The time for an analysis is 57 minutes for one sample, including the extraction step. In conclusion, HS-SPME in combination with GC/MS is an effective method for the determination and quantification of parathion-ethyl and parathion-methyl in biological material.     

Drug testing in blood: validated negative-ion chemical ionization gas chromatographic-mass spectrometric assay for enantioselective measurement of the designer drugs MDEA, MDMA, and MDA and its application to samples from a controlled study with MDMA

BACKGROUND: The enantiomers of the designer drugs 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDEA) differ in their pharmacologic and toxicologic potency. The aim of this study was to develop an assay for measuring these enantiomers in small plasma volumes and to analyze samples from a controlled study with MDMA. METHODS: The analytes were extracted from < or = 0.2 mL of plasma by mixed-mode solid-phase extraction. After derivatization with S-(-)-heptafluorobutyrylprolyl chloride, the resulting diastereomers were separated by gas chromatography (HP-5MS) within 17 min and detected by mass spectrometry in the negative-ion chemical ionization mode. The method was fully validated and applied to samples from a controlled study in which a single dose of racemic MDMA (75 mg) was administered. RESULTS: The derivatized enantiomers were well separated and detected with good sensitivity. The assay was linear (per enantiomer) at 1-50 microg/L for MDA and 5-250 microg/L for MDMA and MDEA. Analytical recovery, accuracy, repeatability, and intermediate precision data were within required limits. Extraction yields were 82.1%-95.3%. In the study samples, concentrations of R-(-)-MDMA significantly exceeded those of S-(+)-MDMA. Their ratios (R vs S) were always >1.0 and increased over time. Concentrations of S-(+)-MDA exceeded those of R-(-)-MDA, their ratios (R vs S) also increasing over time but remaining <1.0. CONCLUSIONS: This assay enables sensitive, reliable, and fast enantioselective measurement of MDA, MDMA, and MDEA in small volumes of plasma. The controlled study data confirm previous findings of MDMA and MDA enantiomer ratios (R vs S) increasing over time after ingestion of racemic MDMA.     

Rapid analysis of metanephrine and normetanephrine in urine by gas chromatography-mass spectrometry.

BACKGROUND: Widely used HPLC methods for quantification of metanephrine and normetanephrine in urine often have long analysis times and are frequently plagued by drug interferences. We describe a gas chromatography-mass spectrometry method designed to overcome these limitations. METHODS: Metanephrine and normetanephrine conjugates were converted to unconjugated metanephrine and normetanephrine by acid hydrolysis. To avoid the rapid decomposition of the deuterated internal standards (metanephrine-d(3) and normetanephrine-d(3)) under hydrolysis conditions, the internal standards were added after hydrolysis. Solid-phase extraction was used to isolate the hydrolyzed metanephrines from urine. Samples were concentrated by evaporation, then derivatized simultaneously with N-methyl-N-(trimethylsilyl)trifluoroacetamide and N-methyl-bis-heptafluoro-butryamide at room temperature. RESULTS: The assay was linear from 25 to 7000 microg/L. The intraassay CVs were < 5 % and the interassay CVs < 12%. Comparison with a routine HPLC method (n = 192) by Deming regression yielded a slope of 1.00 +/- 0.02 microg/L, an intercept of -5.8 +/- 7.8 micro/L, and S(y/x) = 50.6 microg/L for metanephrine and a slope of 0.94 +/- 0.03, intercept of 19 +/- 11 microg/L, and S(y/x) = 60 microg/L for normetanephrine. The correlation coefficients (r) were calculated after log transformation of the data and gave r = 0.97 for metanephrine and r = 0.97 for normetanephrine. Interference from common medications or drug metabolites was seen in <1% of samples. The time between sequential injections was < 7 min. CONCLUSIONS: This new gas chromatography-mass spectrometry assay for total fractionated metanephrines is rapid, compares well with a standard HPLC assay, and avoids most drug interferences that commonly affect HPLC assays for urine metanephrines.     

Continuous improvement of medical test reliability using reference methods and matrix-corrected target values in proficiency testing schemes: Application to glucose assay

The reliability of biological tests is a major issue for patient care in terms of public health that involves high economic stakes. Reference methods, as well as regular external quality assessment schemes (EQAS), are needed to monitor the analytical performance of field methods. However, control material commutability is a major concern to assess method accuracy. To overcome material non-commutability, we investigated the possibility of using lyophilized serum samples together with a limited number of frozen serum samples to assign matrix-corrected target values, taking the example of glucose assays. Trueness of the current glucose assays was first measured against a primary reference method by using human frozen sera. Methods using hexokinase and glucose oxidase with spectroreflectometric detection proved very accurate, with bias ranging between -2.2% and +2.3%. Bias of methods using glucose oxidase with spectrophotometric detection was +4.5%. Matrix-related bias of the lyophilized materials was then determined and ranged from +2.5% to -14.4%. Matrix-corrected target values were assigned and used to assess trueness of 22 sub-peer groups. We demonstrated that matrix-corrected target values can be a valuable tool to assess field method accuracy in large scale surveys where commutable materials are not available in sufficient amount with acceptable costs.     

Evaluation of the BinaxNOW PBP2a assay for the direct detection of methicillin resistance in Staphylococcus aureus from positive blood culture bottles

BinaxNOW® PBP2a rapid immunochromatographic assay is a novel test for the identification of methicillin resistance in Staphylococcus aureus from clinical blood culture samples based on detection of penicillin binding protein 2a. We have evaluated the utility of this assay to do a rapid diagnostic of methicillin susceptibility directly from blood culture bottles after identification of S. aureus in positive bottles by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry. Twenty of 21 methicillin-resistant S. aureus (MRSA) samples from blood cultures were positive on direct immunochromatographic testing (sensitivity 95.24%, 95% confidence interval [CI] 74.13% to 99.75%), whereas 37 methicillin-susceptible S. aureus (MSSA) samples were negative (specificity 100%, 95% CI 88.99% to 99.75%). The combined use of MALDI-TOF mass spectrometry and BinaxNOW® PBP2a test is useful for the rapid identification of both MRSA and MSSA from blood cultures.     

Rapid, accurate, and sensitive fatty acid ethyl ester determination by gas chromatography-mass spectrometry

Background: Fatty acid ethyl esters (FAEEs) are useful markers of ongoing alcohol use and may be associated with alcohol-induced damage to the liver and pancreas. In this article, we describe a novel method for rapid determination of the three major FAEEs found in human plasma. Methods: Internal standard, ethyl heptadecanoate, was added to plasma samples, and FAEEs were isolated by acetone precipitation, hexane lipid extraction, and amino-propyl silica solid phase extraction. FAEEs were quantitated by gas chromatography-mass spectrometry (GC-MS) using a nonpolar dimethylpolysiloxane column. The accuracy, precision, specificity, and sensitivity of the assay were defined from plasma samples from recently drinking and abstinent persons, with and without the addition of FAEEs. Results: Individual FAEE peaks demonstrated excellent resolution. Instrument time was reduced by more than 60%. The lower limit of detection was 5 to 10 nM, and the lower limit of quantitation for each FAEE was 60 nM (for 22 samples with known concentration 60 nM, x +/-SD: 61 +/- 5.7, 57 +/- 5.7, and 57 +/- 5.9 nM, for ethyl palmitate, ethyl oleate, and ethyl stearate, respectively). Instrument precision (coefficient of variance, CV) for these three FAEEs was 0.3%, 0.4%, and 0.7%, respectively. Intra-assay precision (CV) for total FAEEs was less than 7%. FAEEs were absent in 49 samples from abstinent persons. FAEEs were detected in all 76 samples with associated positive blood alcohol levels. Conclusions: Our method of FAEE analysis is rapid and potentially useful in research and clinical studies. FAEE determination using this method is precise, accurate, sensitive, and specific and deserves broader application.