Can an early diagnostic procedure of metformin-associated lactic acidosis in an emergency unit reduce mortality?

OBJECTIVE: Metformin-associated lactic acidosis (MALA) is a rare but serious adverse reaction with a mortality rate of up to 50%. Unfortunately, diagnosis and care management are often delayed. The objective was to assess the impact on the mortality rate and length of hospital stay of a MALA early diagnosis procedure in diabetic patients with metformin at emergency department (ED) admission. METHOD: From 1/7/2012, a new MALA diagnosis procedure (pH, lactate, metformin) was implemented in all diabetic patients with metformin just after their admission to the ED. The pharmacovigilance staff confirmed the MALA cases (defined as pH≤7.35, lactate concentration>5mmol/L) in patients exposed to metformin and after a causality assessment to eliminate other common causes of lactic acidosis. To assess the impact of this new diagnosis procedure, a before-after study was conducted between two groups: a series of cases with intervention (IG; 1/7/2012-30/6/2013) and a control series of past cases without intervention (CG; 1/1/2011-30/6/2012). The main outcome was the relative reduction of mortality rate and length of hospital stay between the two groups. RESULTS: Thirty-four MALA cases were confirmed in 745 subjects admitted with lactic acidosis, (IG: 12; CG: 22). A higher illness severity score in the IG vs. CG was observed: respectively arterial lactate (14.2±6.9 vs. 8.8±5.8mmol/L, P<0.05), arterial bicarbonate (7.8±4.3 vs. 14.3±6.3mmol/L, P<0.05). The median time up to MALA diagnosis was 20.5 (Q1-Q3: 11.3-38.5) minutes for IG and 55.0 (Q1-Q3: 33.0-132.0) minutes for CG. After procedure implementation, the mortality relative risk reduction was 26.7% (95% CI: -84.3%, 70.8%), and especially 54.2% (95% CI: -265.2%, 94.2%) in the ED. There was no difference in the hospital stay duration between the two groups. CONCLUSION: While the results were not significant, the study suggests that the implementation of a MALA early diagnosis procedure in all patients with metformin admitted to an ED tends to decrease mortality, especially for serious MALA cases detected earlier.

Identification and quantification of 35 psychotropic drugs and metabolites in hair by LC-MS/MS: application in forensic toxicology.

Despite a non-invasive sampling, hair samples are generally collected in limited amounts for an obvious esthetic reason. In order to reduce the required quantity of samples, a multianalytes method allowing simultaneous identification and quantification of 35 psychoactive drugs was developed. After incubation of 50 mg of hair in a phosphate buffer pH 5 for one night at room temperature, the substances of interest were extracted by a simple liquid-liquid extraction step, with a dichloromethane/ether mixture (70:30, v/v). After evaporation under a gentle stream of nitrogen and reconstitution in formate buffer (2 mM, pH 3)/acetonitrile (90:10, v/v), twenty microliter were injected into the LC-MS/MS system for a chromatographic run of 29 min using an Atlantis T3 column (150 × 2.1 mm, 3 µm) (Waters Corp, Milford, USA) and a gradient mixture of 2 mM, pH 3.0 ammonium formate, and 2 mM, pH 3.0 ammonium formate/acetonitrile. The data acquisition was performed in scheduled MRM mode. Intra- and inter-day precisions, estimated using the coefficient of variation and relative bias, were lower than 20 % for all concentration levels, except for two compounds. The limits of detection and quantification ranged from 0.5 to 10 pg/mg. After complete validation, this method has been successfully used in several forensic cases, three of which are reported.

Analytical findings of an acute intoxication after inhalation of methoxetamine.

Methoxetamine (MXE) is increasingly used and abused, as it is frequently presented as being safer than ketamine, and legal. Cases of only MXE consumption being associated with the occurrence of seizures are rarely reported. A single MXE intoxication case by inhalation is described concerning a 21-year-old man, not known to be epileptic, who was found collapsed in his bedroom, supposedly after an epileptic seizure. He was transferred to the Emergency Department of the Henri Mondor Hospital, Aurillac, France. He was conscious, but with a sinus bradycardia (48/min) and an ST-segment elevation on the electrocardiogram, and a slightly increased creatine kinase level (270 U/L) and hyponatremia (127 mmol/L). New seizure activity occurred during hospitalization, but the clinical course in the intensive care unit was favorable. Quantitation of MXE in serum and urine using gas chromatography coupled to mass spectrometry (GC-MS) was developed, as well as a liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS) method for the determination of MXE in hair. Limits of detection and quantification were, respectively, 2 and 10 µg/L for the GC-MS method and both 0.5 pg/mg for the LC-MS-MS method. Concentrations of 30 and 408 µg/L were, respectively, measured in serum and urine. Concentrations of 135 and 145 pg/mg were detected in two 2.5 cm hair strands, consistent with one or several consumptions during the 2 ½ months prior to sampling. A sample of the powder consumed was available and also analyzed. This case illustrates the dangers of this drug, which justify its classification as a narcotic in France since August 2013.

Improved liquid chromatography-tandem mass spectrometric method for the determination of ethyl glucuronide concentrations in hair: applications to forensic cases.

Ethyl glucuronide (EtG) is a direct marker of ethanol consumption, and its assay in hair is an efficient tool for chronic alcoholism diagnosis. In 2012, the Society of Hair Testing proposed a new consensus for hair concentrations interpretation, strongly advising the use of analytical methods providing a limit of quantification of less than 3 pg/mg. The present work describes the optimization and validation of a previously developed liquid chromatography-tandem mass spectrometric method in order to comply with this recommendation. The concentration range of this improved method is from 3 to 1,000 pg/mg. Some cases are then described to illustrate the usefulness of hair EtG: a forensic post-mortem case and two cases of suspension of driving licences. Finally, hair samples of some teetotallers (n = 10) have been analyzed, which allowed neither to quantitate nor to detect any trace of EtG.

[Ethylglucuronide assays in urine and hair]. / Dosages urinaire et capillaire de l'éthylglucuronide.

Ethylglucuronide (EtG) is a biomarker of ethanol consumption, and the authors propose an overview of its potential use in clinical or forensic cases. This metabolite presents a higher half-life and is therefore detectable longer than ethanol itself, and allows, in the case of late sampling, the research of acute alcohol abuse in the days prior to urine sampling. Routine testing for urinary EtG can be easily introduced in laboratories since an immunochemical kit is available. However, it is necessary to take into account the cut-off values in order to interpret the results. EtG can also be tested in hair and is a very useful biomarker of chronic alcohol abuse. A recent international consensus has established cut-off values for the interpretation of EtG concentrations in hair. However, hair testing is still limited to specific laboratories as it is not easily implemented in routine, due to a specific sample treatment and to the lack of immunochemical kits.

Suicidal sodium azide intoxication: An analytical challenge based on a rare case.

INTRODUCTION: Intentional absorption of sodium azide is exceptional but remains extremely life-threatening because death rapidly occurs when significant doses are absorbed, either due to the direct effect of sodium azide or an indirect effect due to nitric oxide, cyanide ions or hydrazoic acid production from sodium azide. CASE REPORT: The body of a laboratory assistant, was discovered by his colleagues in the laboratory, seated on a chair located near a digital computer displaying information about sodium azide. Moreover, a half empty 99% sodium azide flask was found near the corpse. The laboratory staff confirmed that the young man was still alive 5h prior to discovery. RESULTS: Postmortem examination did not show any cutaneous signs of injury due to a defensive struggle. Bilateral ungual cyanosis was observed as well as a major cerebral edema and visceral congestion on autopsy. The elevated sodium azide concentration found in the gastric sample and the amount of gastric content allowed to conclude that sodium azide intake was more than 6g which was above the lethal dose, i.e. approximately 1g. Surprisingly, no sodium azide was found either in blood and serum, or in hepatic and renal tissue samplings. However, major concentrations were observed in the gastric contents, bile and urinary samples, as well as in cardiac and cerebral tissues samples. No other toxic element was found. Therefore, the post-mortem findings, the autopsy and the analytical results suggested that the laboratory assistant died after an intentional sodium azide ingestion. CONCLUSION: Sodium azide poisoning by ingestion has to date remained extremely rare and our case highlights the extreme lability of sodium azide as it was absent in the blood, in spite of significant concentrations in stomach content and some tissues. Therefore, the necessity of multiple tissues samples during autopsy should be underlined.

LSD in pubic hair in a fatality.

Lysergic acid diethylamide (LSD) is a potent hallucinogen, active at very low dosage and its determination in body fluids in a forensic context may present some difficulties, even more so in hair. A dedicated liquid chromatography-electrospray-tandem mass spectrometry (LC-ES-MS/MS) assay in hair was used to document the case of a 24-year-old man found dead after a party. Briefly, after a decontamination step, a 50mg sample of the victim's pubic hair was cut into small pieces (<1mm length), and incubated overnight in 3mL of phosphate buffer pH 5 at room temperature. After a liquid-liquid extraction (dichloromethane/ether), the extract was analyzed using a LC-ES-MS/MS method exhibiting a limit of quantification of 0.5pg/mg for LSD. A LSD concentration of 0.66pg/mg of pubic hair was observed. However, this result remains difficult to interpret owing to the concomitant LSD presence in the victim's post mortem blood and urine, the lack of previously reported LSD concentrations in hair, and the absence of data about LSD incorporation and stability in pubic hair.

Determination of dodine in fruit samples by liquid chromatography electrospray ionization-tandem mass spectrometry.

A rapid and sensitive LC/electrospray ionization-MS/MS method has been developed for the determination of dodine in fruit samples. Based on a liquid-liquid extraction of 10 g solid fruit homogenate using an acetone-dichloromethane-hexane mixture and acetate ammonium buffer (pH 4.5), this LC/MS/MS procedure was characterized by recoveries above 50%, with good intra-assay precision (RSD < 13%) and interassay precision (RSD < 18%) for seven different matrixes (apple, apricot, cherry, peach, pear, plum, and quince). This method was validated from 5 to 500 microg/kg according to standard guidelines. Its LOD (1 microg/kg) and LOQ (5 microg/kg) were in accordance with recommendations of the European legislation defined for infant food [maximum residue level (MRL) = 10 microg/kg]. The whole procedure was finally tested on 1022 fruit samples intended for commercialization, both infant food samples and samples not intended in particular for babies. In this study, dodine was detected in 27 samples; none exhibited a concentration higher than the MRL.

Ultra-performance liquid chromatography- tandem mass spectrometry method for the determination of bupropion and its main metabolites in human whole blood.

A selective and sensitive ultra-performance liquid chromatography (UPLC)-electrospray ionization-tandem mass spectrometry (MS) method for simultaneous determination of bupropion and its main metabolites, hydroxybupropion, erythrohydrobupropion, and threohydrobupropion, in human whole blood is presented. The sample preparation consists of cleanup protein precipitation with methanol combined with a solid-phase extraction on Oasis HLB cartridges. Analytes were separated on a Waters Acquity UPLC((R)) BEH phenyl column using a binary mobile phase consisting of ammonium formate buffer (2 mM, pH 4) and acetonitrile. Detection was performed on a Waters Acquity UPLC system coupled to a Quattro Premier triple-quadrupole MS in positive ion selected reaction monitoring. Internal standards were bupropion-d(9) and hydroxybupropion-d(6). Linearity was from 5 to 1000 ng/mL for bupropion and from 10 to 2000 ng/mL for metabolites. Accuracy profiles (80-120%), precision (< 15%), and limits of detection (1 ng/mL for bupropion and 2 ng/mL for metabolites) were also evaluated and responded to all criteria of validation. The aim of this study was to compare this presented method with a previously described method developed on a classic liquid chromatography-tandem MS system.

Stability of ribavirin concentrations depending on the type of blood collection tube and preanalytical conditions.

Ribavirin pharmacokinetic and exposure effect trials based on either plasma or serum concentrations have yielded diverging results. This study aimed to compare ribavirin concentrations in serum and plasma and to investigate the influence of blood collection and preanalytical conditions on ribavirin concentration stability. Blood samples from patients with hepatitis virus C and receiving ribavirin were collected in plain (dry) tubes, in tubes containing ethylenediaminetetra-acetic acid or lithium-heparinate, in Type II Serum Separating Tubes with clot activator, or Type II lithium heparinate Plasma Separating Tubes. Different time and temperature conditions were tested before and after blood centrifugation. Ribavirin was determined using liquid chromatography-dual mass spectroscopy. Multiple-way analysis of variance was used for statistical analyses. Ribavirin concentrations showed a higher interlaboratory variability in serum than in plasma. Results were fairly stable over 2 hours in whole blood collected in dry or ethylenediaminetetra-acetic acid tubes and very stable up to 24 hours in serum or plasma kept in gel-containing tubes after immediate centrifugation. When Plasma Separating II gel tubes were kept at +4 degrees C or at ambient temperature for up to 24 hours before centrifugation, ribavirin concentrations decreased by 1% to 8% and 12% to 18%, respectively. These results suggest that blood samples should be collected in gel-containing tubes and centrifuged immediately, after which the tubes can be kept at ambient temperature for the next 24 hours. In case of clinical constraints, Plasma Separating II gel tubes can be kept at +4 degrees C for a maximum of 2 hours before centrifugation with limited impact on the measured concentrations.

Screening of pesticides in blood with liquid chromatography-linear ion trap mass spectrometry.

In clinical or forensic toxicology, general unknown screening procedures are used to identify as many xenobiotics as possible, belonging to numerous chemical classes. We present here a general unknown screening procedure based on liquid chromatography coupled with use of a single linear ion trap mass spectrometer, and focus on the identification of pesticides and/or metabolites in whole blood. After solid-phase extraction (SPE), the compounds of interest were separated using a reversed-phase column and identified by the mass spectrometer operated first in the full-scan mass spectrometry (MS) mode, in the positive and negative polarities, followed by MS(2) and MS(3) scanning of ions selected in data-dependent acquisition. The total scan time was 2.45 s. Two mass spectral libraries (MS(2) and MS(3)), each of 450 spectra, were created for the 320 pesticides and metabolites detected after injection of pure solutions. Robustness of the spectra and matrix effects were studied and were satisfactory for the present application. Detection limits for the 320 compounds were studied by extracting 1 mL spiked blood at concentrations between 10 microg/L and 10 mg/L. If necessary, it was possible to decrease the detection limits of some compounds by 10-100-fold by scanning MS(2) in only one polarity, owing to a shorter total scan time. However, at the same time, the detection specificity decreased as no confirmation could be recorded in the following MS(3) scan and no information could be registered in the other polarity. So, in these rare cases, confirmation by another method was required.

General unknown screening procedure for the characterization of human drug metabolites in forensic toxicology: applications and constraints.

LC coupled to single (LC-MS) and tandem (LC-MS/MS) mass spectrometry is recognized as the most powerful analytical tools for metabolic studies in drug discovery. In this article, we describe five cases illustrating the utility of screening xenobiotic metabolites in routine analysis of forensic samples using LC-MS/MS. Analyses were performed using a previously published LC-MS/MS general unknown screening (GUS) procedure developed using a hybrid linear IT-tandem mass spectrometer. In each of the cases presented, the presence of metabolites of xenobiotics was suspected after analyzing urine samples. In two cases, the parent drug was also detected and the metabolites were merely useful to confirm drug intake, but in three other cases, metabolite detection was of actual forensic interest. The presented results indicate that: (i) the GUS procedure developed is useful to detect a large variety of drug metabolites, which would have been hardly detected using targeted methods in the context of clinical or forensic toxicology; (ii) metabolite structure can generally be inferred from their "enhanced" product ion scan spectra; and (iii) structure confirmation can be achieved through in vitro metabolic experiments or through the analysis of urine samples from individuals taking the parent drug.

Determination of ethyl-glucuronide in hair for heavy drinking detection using liquid chromatography-tandem mass spectrometry following solid-phase extraction.

The detection of ethyl-beta-D-6-glucuronide (EtG), a stable phase II metabolite of ethanol, is of interest in both clinical and forensic contexts with the aim of monitoring alcohol abuse. We present a liquid chromatography-electrospray ionisation-tandem mass spectrometry method for the detection and quantification of EtG in hair. Thirty milligrams of washed and cut hair were cleaned up using solid-phase extraction graphite cartridges. Separation was then performed using an Uptisphere-3SI column, and the detection was operated in the negative mode. After validation, the method was applied to hair samples taken from four fatalities (F) with documented excessive drinking habits, 12 heavy drinkers (HD) and seven social drinkers (SD). The method exhibits limits of detection and quantification of 4 and 10 pg/mg, respectively. Intra- and inter-assay standard deviation and relative bias were less than 20% over the calibrating range (10 to 3,000 pg/mg). EtG hair concentrations in SD were <10 pg/mg and >50 pg/mg for F and HD (range, 54 to 497 pg/mg). The present assay appears convenient to carry out owing to the very small quantity of hair samples required to determine an effective heavy alcohol consumption (EtG hair concentration >50 pg/mg).

Pitfalls and prevention strategies for liquid chromatography-tandem mass spectrometry in the selected reaction-monitoring mode for drug analysis.

BACKGROUND: We observed cases of false-positive results with the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Different LC-MS/MS techniques that use the selected reaction-monitoring mode, routinely employed for the analysis and quantification of drugs and toxic compounds in biological matrices, were involved in the false-positive and potentially false-positive results obtained. We sought to analyze the causes of and solutions to this problem. METHODS: We used a previously reported LC-MS/MS general unknown screening method, as well as manual spectral investigation in 1 case, to perform verification and identification of interfering compounds. RESULTS: We observed that false-positive results involved: a metabolite of zolpidem that might have been mistaken for lysergic acid diethylamide, benzoylecgonine mistaken for atropine, and clomipramine and 3 phenothiazines that share several common ion transitions. CONCLUSIONS: To prevent problems such as those we experienced, we recommend the use of stable-isotope internal standards when possible, relative retention times, 2 transitions or more per compound when possible, and acceptable relative abundance ratios between transitions, with an experience-based tolerance of +/-15% for transitions with a relative abundance >10% and with an extension to +/-25% for transitions <10% when the concentration is at the limit of quantification. A powerful general unknown screening procedure can help to confirm suspected interferences. Our results indicate that the specificity of screening procedures is questionable for LC-MS/MS analyses performed in the selected reaction-monitoring mode and involving a large number of compounds with only 1 transition per compound.

Identification of acepromazine in hair: an illustration of the difficulties encountered in investigating drug-facilitated crimes.

After a drug-facilitated sexual assault (DFSA), a woman was found in a drowsy state at home. She remembered having drunk an unknown beverage by the accused. Blood samples (collected 8 hours after the DFSA), two glasses, and a teaspoon seized by the police were analyzed. Acepromazine, a phenothiazine tranquilizer used in human and veterinary medicine, was detected in the residue of one of the glasses. In spite of acepromazine absence in the victim's blood, the possible use of acepromazine in the DFSA was reported to the police. Two weeks later, a suspect admitted having orally administered acepromazine to the victim. Using a liquid chromatography-tandem mass spectrometry method, this compound was subsequently detected (31 pg/mg) in a sample of the victim's hair collected a month and a half after the DFSA. A potential short elimination half-life in humans and/or the well-known in vitro degradation of acepromazine could explain the negative blood result. DFSA toxicological investigations are challenging and can be complicated when a rather unusual substance is concerned. In particular, special care should be taken when interpreting the results, taking into account elimination and/or instability data, when available.

A fatal case of bupropion (Zyban) overdose.

A sensitive and reproducible method for the identification and the quantitative determination of bupropion (BUP) and its major metabolites, hydroxybupropion (OH-BUP) and threohydrobupropion (T-BUP), was developed in blood and urine. The three compounds were extracted with a solid-phase extraction procedure followed by LC-ESI-MS-MS separation and quantification using decadeuterated lidocaine as internal standard. BUP and its metabolites were satisfactorily identified by multiple reactions monitoring detection. The limits of detection and quantification were determined at 5 and 10 microg/L, respectively, for each analyte. The intraday and interday coefficients of variability were lower than 11.9% for BUP and its metabolites. This method was applied to the forensic case of a 35-year-old male who died after a suspected ingestion of 30 slow-release tablets of Zyban. As samplings were performed at least 72 h after the drug intake, BUP had disappeared from blood, but OH-BUP and T-BUP were present at the concentrations of 5.8 and 30.4 mg/L, respectively. In urine, concentrations ranged from 42.9 mg/L for BUP to 617 mg/L for T-BUP. These results agree with the hypothesis of a successful suicide attempt.

Screening of drugs and toxic compounds with liquid chromatography-linear ion trap tandem mass spectrometry.

BACKGROUND: In clinical and forensic toxicology, general unknown screening is used to detect and identify exogenous compounds. In this study, we aimed to develop a comprehensive general unknown screening method based on liquid chromatography coupled with a hybrid triple-quadrupole linear ion trap mass spectrometer. METHODS: After solid-phase extraction, separation was performed using gradient reversed-phase chromatography. The mass spectrometer was operated in the information-dependent acquisition mode, switching between a survey scan acquired in the Enhanced Mass Spectrometry mode with dynamic subtraction of background noise and a dependent scan obtained in the enhanced product ion scan mode. The complete cycle time was 1.36 s. A library of 1000 enhanced product ion-tandem mass spectrometry spectra in positive mode and 250 in negative mode, generated using 3 alternated collision tensions during each scan, was created by injecting pure solutions of drugs and toxic compounds. RESULTS: Comparison with HPLC-diode array detection and gas chromatography-mass spectrometry for the analysis of 36 clinical samples showed that linear ion trap tandem mass spectrometry could identify most of the compounds (94% of the total). Some compounds were detected only by 1 of the other 2 techniques. Specific clinical cases highlighted the advantages and limitations of the method. CONCLUSION: A unique combination of new operating modes provided by hybrid triple-quadrupole linear ion trap mass spectrometers and new software features allowed development of a comprehensive and efficient method for the general unknown screening of drugs and toxic compounds in blood or urine.

Liquid chromatography-tandem mass spectrometry for detection of low concentrations of 21 benzodiazepines, metabolites, and analogs in urine: method with forensic applications.

BACKGROUND: Commonly used methods for detecting benzodiazepines (BZPs) and BZP-like substances, such as zolpidem and zopiclone, may not detect low concentrations of these drugs. We developed a liquid chromatographic-tandem mass spectrometric method for identifying these drugs and their relevant metabolites. METHODS: We extracted BZPs from urine by solid-phase extraction with a mixed-mode phase (OASIS HLB cartridges). Chromatographic separation was performed with a Waters XTerra MS C18 [150 x 2.1 mm (i.d.); bead size, 5 microm] reversed-phase column with deuterated analogs of the analytes as internal standards (IS). Detection was performed with a triple-quadruple mass spectrometer that monitored 2 specific transitions per compound in the electrospray, positive-ion selected-reaction monitoring mode. We tested this technique on urine samples from 12 healthy volunteers and 1 forensic sample obtained in a case of alleged drug-facilitated sexual assault. RESULTS: Chromatographic separation was achieved within 18 min. The linear dynamic ranges extended from 0.02 or 0.1 microg/L (depending on the drug or metabolite) to 50 microg/L. Extraction recovery (range) was 77%-110%. Limits of detection were < or = 0.05 microg/L. No ion suppression was seen except for alprazolam, for which baseline decreased by almost 20%. In the forensic urine sample, the method detected alprazolam (3.5 microg/L) and its characteristic metabolite, alpha-hydroxyalprazolam (0.17 microg/L). CONCLUSION: This method measured low concentrations of BZPs and BZP-like substances and might be useful for analyses of urine in suspected drug-facilitated sexual assault cases.

Post-mortem redistribution of three beta-blockers in the rabbit.

To consider the role of the physico-chemical properties of drugs in their post-mortem redistribution, we designed the present study to investigate the influence of lipophilicity using an experimental rabbit model. Three beta-blockers (BB), atenolol, metoprolol and propranolol, with a similar dissociation constant (pK (a)) and increasing partition coefficient (K (p)) were administered intravenously to 18 rabbits. One hour after the last administration, the animals were killed by thiopental injection and placed in a supine position at room temperature. Autopsies were performed at 0, 2, 6, 12, 24 and 48 h post-mortem. Concentrations of the three BB were determined in fluids (right and left cardiac blood, peripheral blood, urine, bile, stomach content, vitreous humour) and tissues (cardiac muscle, lungs, liver, brain, diaphragm, iliopsoas muscle) using a previously published, validated liquid chromatography-electrospray-mass spectrometry method. Our results show that lipophilicity influences post-mortem redistribution of the molecules in a certain number of anatomical sites such as the stomach, lungs, cardiac muscle, cardiac blood or liver, but does not appear to intervene in other sites such as the brain or the vitreous humour.