This application note describes the rapid cleanup of serum samples and detection of over 100 drugs using On-line Solid Phase Extraction (SPE) LC-MS/MS screening and confirmation. The LC-MS/MS was operated in Multiple Reaction Mode (MRM) for detection. Dependent MS/MS spectra were acquired in the Enhanced Product Ion (EPI) mode after being triggered from a Scheduled MRMTM Pro Algorithm Information Dependent Acquisition (IDA) survey scan. Combining MRM and product ion spectral acquisition allows for compound identification with highest confidence based on mass spectral library matching. The automated workflow monitors large panels of analytes, detecting and quantifying these compounds in a single run.

Background: Gamma-hydroxybutyric acid (GHB) has become one of the most dangerous illicit drugs of abuse today. It is used as a recreational and date rape drug because of its depressant effect on the central nervous system, which may cause euphoria, amnesia, respiratory arrest, and coma. There is an urgent need for a simple, easy-to-use assay for GHB determination in urine and blood. In this article, a rapid enzymatic assay adapted to clinical chemistry analyzers for the detection of GHB is presented. Methods: The described GHB enzymatic assay is based on a recombinant GHB dehydrogenase. The full validation of the assay was performed on a Konelab 30 analyzer (Thermo Fisher Scientific). Results: The analytical sensitivity was <1.5 mg/L, whereas the functional sensitivity was 4.5 mg/L in serum and 2.8 mg/L in urine. The total imprecision coefficient of variation (CV) was <9.8% in serum and <7.9% in urine. The within-run imprecision showed a CV of <3.8% in serum and <4.6% in urine. The assay was linear within the range 5–250 mg/L. Mean recoveries were 109% in serum and 105% in urine. No cross-reactivity was observed for tested GHB analogues and precursors. Comparison of GHB-positive samples showed an excellent correlation with ion chromatography, gas chromatography–mass spectrometry, and liquid chromatography associated to tandem mass spectrometry. Except for ethanol, no substantial interference from serum constituents and some drugs was observed. Conclusions: This automated GHB assay is fully quantitative and allows the accurate measurement of GHB in serum and urine. It can be used as a rapid screening assay for the determination of GHB in intoxicated or overdosed patients.

A fully automated screening using liquid chromatography–mass spectrometric method applying data-dependent acquisition was developed to identify toxicologically relevant substances in serum and urine. A library including more than 405 spectra of about 365 compounds (main drugs and important metabolites) was established. An easy to use program was created to automate and accelerate library search. Drugs were identified based on their relative retention times, molecular ions and fragment ions. Limits of detection were tested with 100 of the 365 compounds the majority of these were lower than 100μg/l (67%). The developed LC–MS–MS system seems to be a valuable alternative to other general unknown screening methods allowing fast and specific identification of drugs in serum and urine samples.

Gamma hydroxybutyric acid (GHB) is a regulated therapeutic drug, which naturally occurs in mammalian brain tissues as an intermediate of the GABA (gamma aminobutyric acid) neurotransmitter metabolism. The increasing misuse of GHB as a narcotic or abusing drug in recent years calls for the development of a simple and rapid screening method as an alternative to the currently available, technically demanding diagnostic methods. We have developed a rapid enzymatic assay based on the GHB dehydrogenase of Ralstonia eutropha. The enzyme is expressed as a recombinant protein in Escherichia coli and characterized in terms of reaction mechanism and kinetic parameters for the catalysis of conversion of GHB into succinic semialdehyde (SSA). The concomitant NADH production enables spectrophotometric monitoring of the reaction and the quantification of GHB in physiological fluids depending on initial velocities. We have tested a panel of twelve serum and urine samples containing GHB concentrations from 0.0 to 2.1 mmol/L. GHB dehydrogenase activity obeys a non classical bi bi ping pong mechanism exhibiting substrate inhibition by NAD+. With an optimal NAD+ concentration of 3.7 mmol/L in the reaction, the enzyme yields a K(M) of 1.0 mmol/L for GHB and a Vmax of 3.37 mmol/min/mg. The assay shows a linear standard curve from 0.1 to at least 1 mmol/L of GHB. Spiking experiments result in mean recoveries of 92% for urine and 114% for serum, respectively. The comparison to an ion chromatographic reference method exhibits a mean difference of 10% divergence from the target values in urine and 9% in serum, respectively.

Introduction: Phenytoin is a drug used for the treatment of different types of seizures. Its variable pharmacokinetics, mainly a consequence of variable bioavailability, saturable protein binding and saturable hepatic metabolism, predisposes the drug to therapeutic drug monitoring. Several methods to analyze the drug in serum exist with immunoassays being the method of choice for routine measurements. Immunoassays are specific and sensitive, but cross-reactivity, possibly leading to erroneous serum levels, is a concern. We report a patient with falsely undetectable phenytoin serum levels.Case report: This 73-year old woman was treated with intravenous phenytoin due to epilepsia partialis continua in the context of a bilateral cerebrovascular insult: Anamnestically, a chronic lymphatic leukemia was known. In this patient, serum phenytoin levels became only detectable by the particle enhanced turbidimetric inhibition immunoassay used after precipitation of serum proteins. Protein electrophoresis revealed a monoclonal immunoglobulin, identified as IgM lambda. With other methods such as HPLC and fluorescence depolarization immunoassay, phenytoin was detectable.Conclusion: We propose interference between the monoclonal IgM lambda and/or other serum proteins and the particle-enhanced turbidimetric inhibition immunoassay, rendering phenytoin falsely undetectable in samples of this patient. In such patients, alternative methods such as HPLC should be used to prevent dosage errors. (c) 2007 Elsevier B.V. All rights reserved.

For forensic and clinical toxicologic purposes, cannabis consumption is screened using easy-to-handle immunoassays. The sensitivity and specificity of these immunoassays have not yet been established in samples from volunteers receiving oral synthetic tetrahydrocannabinol or cannabis extracts using liquid chromatography-mass spectrometry/mass spectrometry as the reference method. Urine samples were collected in an open, randomized, single-center, three-period crossover study including 18 healthy male volunteers given either 20 mg synthetic tetrahydrocannabinol (Marinol) as a control substance or five different types of Cannabis sativa extracts.

A method for the determination of γ-hydroxybutyric acid (GHB) in urine and serum samples with capillary electrophoresis using capacitively coupled contactless conductivity detection (CE-C4D) was developed. The optimized separation buffer consisting of 20mM of arginine, 10mM of maleic acid and 30μM of cetyltrimethylammonium bromide (CTAB) contained 5mM vancomycin to facilitate the separation of γ-hydroxybutyric acid from β-hydroxybutyric acid (BHB), which is also present in clinical samples. The detection limits in the clinical samples were found to be about 2μg/ml, which is well below the required sensitivity for the recognition of overdosage and adequate for the determination of endogenous concentrations in urine. The determination of GHB in both types of samples was carried out directly after a fourfold dilution without requiring any derivatization or extraction procedures.

A novel high-performance liquid chromatographic separation method with tandem-mass spectrometry detection was developed for the simultaneous determination of Δ9-tetrahydrocannabinol (THC) and its major metabolites 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) as well as the components cannabidiol (CBD) and cannabinol (CBN) in human EDTA-plasma and urine. Run time was 25min. Lower limit of quantification was 0.2ng/ml. The coefficients of variation of all inter- and intra-assay determinations were between 1.3 and 15.5%. The method was successfully applied to the determination of cannabinoids in human plasma and human urine after administration of Δ9-tetrahydrocannabinol or Cannabis sativa extracts.

BACKGROUND:The objective of this prospective study was to assess the medical and economic long-term effects of using B-type natriuretic peptide (BNP) concentrations in the management of patients with acute dyspnea.METHODS:We performed follow-up analysis of the B-Type Natriuretic Peptide for Acute Shortness of Breath Evaluation, a randomized study including 452 patients who presented to the emergency department with acute dyspnea. Participants were randomly assigned to a diagnostic strategy involving the rapid measurement of BNP concentrations (n = 225) or standard assessment (n = 227). Mortality was assessed at 720 days, morbidity and economic data at 360 days.RESULTS:BNP testing induced several important changes in initial patient management, including a reduction in the initial hospital admission rate, the use of intensive care, and initial time to discharge. At 720 days, 172 deaths had occurred. Cumulative all-cause 720-day mortality was not different between the BNP group (37%) and the control group (36%, P = 0.6). Morbidity as reflected by days spent in-hospital at 360 days was significantly lower in the BNP group [median 12 days ([interquartile range 2-28 days)] compared with the control group [median 16 (7-32)] days, P = 0.025]. Functional status was similar in both groups. Economic outcome as quantified by total treatment cost at 360 days was significantly improved in the BNP group (mean 10,144 dollars vs 12,748 dollars in the control group, P = 0.008).CONCLUSIONS:Rapid BNP testing in patients with acute dyspnea has no effect on long-term mortality. However, morbidity as quantified by days spent in-hospital and economic outcome are still improved at 360 days.

de documenter l'évolution d'une néphropathie.

An immunoassay of sirolimus based on the microparticle enzyme immunoassay (MEIA) principle was evaluated on 105 whole blood samples (EDTA) drawn from a population of renal (n = 38) and hepatic or heart (n = 3) transplant patients. Each blood sample was analyzed simultaneously by MEIA and by a liquid chromatography mass spectrometry (LC-MS) method. The statistical analysis according to Passing-Bablok (x= LCMS, y= mean of two measurements of the same samples on IMx) produced the following results: Spearman r value = 0.9651, y(MEIA) = 0.99x (LCMS) - 0.26 microg/l. The analytical performance of the MEIA method showed a CV < or = 10% and a lower limit of quantification of 1.5 microg/l, which are acceptable for routine clinical monitoring. In conclusion, the MEIA method has shown robust, stable and reproducible features with an excellent correlation with the LC-MS method.

We report the case of attempted suicide with amlodipine, chlorthalidone and mefenamic acid and subsequent medical intensive care measures which resulted in total recovery of a 42-year-old male. After admission to the medical intensive care unit the intoxicated patient was deeply hypotensive and needed fluid replacement, dobutamine and norepinephrine. Additionally insulin and calcium gluconate were given. Since hypotension persisted and the patient developed oliguria, terlipressin was applied and finally showed an effect on blood pressure and on urinary output. A volume overload of 7 L in the first 24 h resulted in a pulmonary edema. The patient was started on non-invasive ventilation with continuous positive airway pressure (CPAP) and frusemide was added to the therapy with good success. Quantitative determination of amlodipine in plasma samples was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The highest amlodipine concentrations was measured in the plasma sample collected approximately 8 h after ingestion of the drug, and was 393 microg/L. Four days later, it was possible to stop the treatment with catecholamines, at that time the amlodipine plasma concentration had declined to 132 microg/L, still tenfold higher than therapeutic (5-18 microg/L). Elimination half-life of amlodipine is approximately 55 h. After 6 days in the intensive care unit the patient was transferred to psychiatric treatment. Intensive care management and plasma levels in this intoxication case are compared to data from literature on other cases.

Zusammenfassung Die Analyse von spezifischen Proteinen im zweiten Morgenurin, bezogen auf den Kreatiningehalt der Probe, erlaubt heute nicht nur den Nachweis oder den Ausschluss von Nierenerkrankungen, sondern darüber hinaus auch die Differenzierung und Verlaufskontrolle von Nephropathien. Störungen lassen sich aufgrund ihres Markerproteinprofils in solche mit hauptsächlich glomerulärem oder tubulärem Anteil und zusätzlich in weitere Untergruppen einteilen. Im Zusammenhang mit den Teststreifenresultaten kann die Quelle einer Blutung mit spezifischen Quotienten näher eingegrenzt und Kontaminationen können von tatsächlichen renalen Proteinurien unterschieden werden. Eine Plausibilitätsprüfung und Interpretation der erhaltenen Ergebnisse ist unbedingt erforderlich. Da eineVielzahl von Regeln überprüft werden muss, ist eine Berechnung und Darstellung der Ergebnisse nur mit Hilfe von wissensbasierten Systemen in Kombination mit einer grafischen Befunddarstellung sinnvoll.

OBJECTIVE:Co-medication unknown to the treating physician, including self-medication, may compromise drug safety by increasing the risk of duplicate therapy, drug interactions and adverse drug reactions that are not recognised as such. The aim of the current study was to estimate exposure to unknown co-medication during hospitalisation by performing an analytical screening for a broad range of drugs and drug classes in urine of patients admitted to a general internal medicine ward.METHODS:Urine samples of 44 patients were analysed with REMEDiHS (high-performance liquid chromatography) and six different immunoassays. Positive results were compared with drug history and documented drug prescription. If appropriate, gas chromatographic-mass spectrometric confirmatory analyses were performed on drugs classified at least once as possible unknown co-medication.RESULTS:Nine (20%) of the patients tested positive for a compound detected by two independent analytical methods and 18 (41%) for a compound detected by at least one analytical method. Unknown co-medication consisted mostly of analgesics, benzodiazepines or ranitidine.CONCLUSION:At least one in five patients exhibits at least once during hospitalisation exposure to drugs not documented in the patient record, which may compromise patient safety.