Marek Dziadosz

Scheduled multiple reaction monitoring algorithm as a way to analyse new designer drugs combined with synthetic cannabinoids in human serum with liquid chromatography–tandem mass spectrometry

Here, we describe the development and application of a liquid chromatography-tandem mass spectrometry method with positive electrospray ionisation and scheduled multiple reaction monitoring algorithm (s-MRM) to analyse synthetic cannabinoids (SC) combined with new designer drugs (NDD) in human serum. A Luna 5μm C18 (2) 100A, 150mm×2mm analytical column and a mobile phase consisted of A (H2O/methanol=95/5, v/v) and B (H2O/methanol=3/97, v/v) - both with 10mM ammonium acetate and 0.1% acetic acid (pH=3.2), were used for the separation. A binary flow pumping mode with a total flow rate of 0.400mL/min was used. A single sample extraction with 1-chlorobutane for both substance groups was performed. Acceptable linearity in the validated calibration ranges of 0.05-1ng/mL for SC and 1-50ng/mL for NDD was achieved. The limit of detection was not greater than 0.02/0.40ng/mL and the limit of quantification not greater than 0.05/0.50ng/mL for SC/NDD respectively. The presented study revealed that this method is a very effective way for sensitive SC and NDD identification in human serum and has useful application in hospitals, therapy centres and forensic psychiatric centres. S-MRM ensures a method upgrade with a smaller loss of sensitivity, precision and accuracy in comparison to traditional MRM methods. Also addition of new SC and NDD can be performed in the future.

Small molecule adduct formation with the components of the mobile phase as a way to analyse valproic acid in human serum with liquid chromatography-tandem mass spectrometry

A valproic acid (VPA) LC-MS/MS analytical method using analyte adduct formation was developed and validated in human serum. The fragmentation of the sodium acetate adduct (mass transition: 225/143) and acetic acid adduct (mass transition: 203/143) were used as the target and qualifier mass transition, respectively. A Luna 5 μm C18 (2) 100 A, 150 mm×2 mm analytical column and a mobile phase consisting of A (H2O/methanol=95/5, v/v) and B (H2O/methanol=3/97, v/v), both with 10mM ammonium acetate and 0.1% acetic acid (pH=3.2) were used. A binary flow pumping mode with a total flow rate of 0.4 mL/min was applied. Protein precipitation with 1 mL of the mobile phase B was used as sample preparation. The calculated limit of detection/quantification was 0.45/1.0 μg/mL and the inter-/intra-day precision was <6%. The application of a deuterated internal standard resulted in a good adduct formation reproducibility. The strategy applied made the VPA LC-MS/MS analysis in human serum on the basis of two mass transitions possible. Therefore, it is an interesting alternative for the VPA pseudo multiple reaction monitoring methods (mass transition 143/143) and a proof that the developed strategy is also useful for the analysis of compounds which do not produce any stable ion fragments detectable by tandem mass spectrometry.

Postmortem concentration distribution in fatal cases involving the synthetic opioid U-47700

Dear Editor, U-47700 is a synthetic drug from the group of new psychoactive substances, used illicitly by recreational opioid users. Animal models revealed that U-47700 is around 7.5 times more potent in binding to opioid receptors than morphine. Its high potency might lead to numerous lethal intoxications, like reported recently after U-47700 consumption [1–4]. Especially, a combined application of U-47700 with other opioids has to be classified as dangerous [5]. Like already pointed out, there is insufficient information about U-47700 concentration in biological samples [6]. Therefore, with this letter, we wanted to present our data concerning U-47700 concentration distribution in postmortem specimens collected after two lethal intoxications. In fatalities investigated, both decedents were found dead on a bed. Because U-47700 was characterized as a selective and strong opioid receptor agonist, generally leaving uncharacteristic findings, the autopsies performed could not reveal the cause of death. Since cerebral/lung oedema and a full urinary bladder were found, a possible intoxication was discussed. The postmortem specimens collected by forensic pathologists underwent blood alcohol analysis, broad toxicological examinations with general unknown analyses, and appropriate confirmations of targeted drugs. No relevant concentrations of other substances than U-47700 could be detected (Table 1). Appropriate quantifications in postmortem specimens, performed on the basis of liquidliquid extraction with 1-chlorobutane, standard addition (with spikes added at three levels), fentanyl-D5 applied as internal standard, and liquid chromatography-tandem mass spectrometry, revealed U-47700 concentrations as presented in Table 1. As expected, femoral blood concentrations were lower than the urine/heart blood concentrations and can be defined as high when compared with toxic morphine levels. They can also be classified as lower than the concentration discussed by Elliott et al. [1] but higher than the data presented by Mclntyre et al./Mohr et al. [2, 3]. Regarding all the circumstances, the cause of death could be explained by the U-47700 consumption in both cases. It is well known that the interpretation of postmortem drug concentration is a big challenge [7]. Scarcity of information on reference concentrations in postmortem matrices makes the interpretation even more problematic. Since the analyses in cases discussed were performed in a variety of postmortem specimens, the concentration distribution data provided can be * Marek Dziadosz analytiker@chemist.com; Dziadosz.Marek@mh-hannover.de

Applicability of Adduct Detection in Liquid Chromatography–Tandem Mass Spectrometry

To investigate the process of analyte adduct formation with the components of the mobile phase in the negative electrospray ionization mode and to answer the question if the strategy of adduct identification/fragmentation is applicable for a bigger number of drugs, which meet the appropriate ionization conditions and compare the detection of appropriate analyte adducts to deprotonated analytes with tandem mass spectrometry, ten drugs containing carboxyl groups capable of negative electrospray ionization were chosen for appropriate experiments. The continuous infusion of captopril, fenbufen, gabapentin, ibuprofen, ketoprofen, naproxen, ramipril, salicylic acid, tiaprofenic acid, and 11-nor-9-carboxy-delta-9-tetrahydrocannabinol made the adduct identification/fragmentation experiments possible. The results achieved show that the adduct formation/fragmentation process can be used for LC–MS/MS analysis of drugs that meet the appropriate ionization conditions like the presence of a carboxyl group. It was also demonstrated that signal intensities achieved by the fragmentation of deprotonated drugs are in most cases higher than the signals connected with the fragmentation of appropriate adducts.

γ-Hydroxybutyrate analysis in human serum with liquid chromatography–tandem mass spectrometry on the basis of MS3 mass transition

An LC-MS/MS/MS GHB quantification method in human serum supported by the adduct formation process with the components of the mobile phase was developed and validated. The continuous infusion of GHB made the identification of a GHB MS3 mass transition possible (185/103/85). A Luna 5 μm C18 (2) 100Å, 150 mm × 2 mm analytical column and the elution with a programmed flow of the mobile phase consisting of 10% A (H₂O/methanol=95/5, v/v) and 90% B (H₂O/methanol=3/97, v/v), both with 10 mM ammonium acetate and 0.1% acetic acid (pH=3.2) were used. The analytical sample preparation consisted of protein precipitation with 1 mL of the mobile phase B. The calculated limit of detection/quantification was 0.28/0.96 μg/mL, respectively. The study presented demonstrated that the process of analyte adduct formation with the components of the mobile phase can be used successfully to generate MS3 transitions in the case of very small molecules which cannot be detected in this way by the application of conventional analytical strategies.

The application of multiple analyte adduct formation in the LC–MS3 analysis of valproic acid in human serum

LC-MS using electrospray ionisation (negative ion mode) and low-energy collision-induced dissociation tandem mass spectrometric (CID-MS/MS) analysis, together with the multiple analyte adduct formation with the components of the mobile phase, were applied to analyse valproic acid in human serum with LC-MS3. The CID-fragmentation of the precursor analyte adduct [M+2CH3COONa-H]- was applied in the method validation (307.1/225.1/143.0). Chromatographic separation was performed with a Luna 5μm C18 (2) 100A, 150mm×2mm column and the elution with a mobile phase consisting of A (H2O/methanol=95/5, v/v) and B (H2O/methanol=3/97, v/v), both with 10mM ammonium acetate and 0.1% acetic acid. A binary flow pumping mode with a total flow rate of 0.400mL/min was used. The calculated limit of detection/quantification of the method calibrated in the range of 10-200μg/mL was 0.31/1.0μg/mL. The sample preparation based on protein precipitation with 1mL of H2O/methanol solution (3/97, v/v) with 10mM sodium acetate and 100mM acetic acid. On the basis of the experiments performed could be demonstrated, that multiple analyte adduct formation can be applied to generate MS3 quantitation of analytes with problematic fragmentation. The presented new strategy makes the analysis of small drugs, which do not produce any stable product ions at all, on the basis of LC-MS3 possible.

Direct analysis of ethylene glycol in human serum on the basis of analyte adduct formation and liquid chromatography–tandem mass spectrometry

The aim of this work was to develop a fast, cost-effective and time-saving liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical method for the analysis of ethylene glycol (EG) in human serum. For these purposes, the formation/fragmentation of an EG adduct ion with sodium and sodium acetate was applied in the positive electrospray mode for signal detection. Adduct identification was performed with appropriate infusion experiments based on analyte solutions prepared in different concentrations. Corresponding analyte adduct ions and adduct ion fragments could be identified both for EG and the deuterated internal standard (EG-D4). Protein precipitation was used as sample preparation. The analysis of the supernatant was performed with a Luna 5μm C18 (2) 100A, 150mm×2mm analytical column and a mobile phase consisting of 95% A (H2O/methanol=95/5, v/v) and 5% B (H2O/methanol=3/97, v/v), both with 10mmolL-1 ammonium acetate and 0.1% acetic acid. Method linearity was examined in the range of 100-4000μg/mL and the calculated limit of detection/quantification was 35/98μg/mL. However, on the basis of the signal to noise ratio, quantification was recommended at a limit of 300μg/mL. Additionally, the examined precision, accuracy, stability, selectivity and matrix effect demonstrated that the method is a practicable alternative for EG quantification in human serum. In comparison to other methods based on liquid chromatography, the strategy presented made for the first time the EG analysis without analyte derivatisation possible.

Letter to the Editor—Consumption of Levamisole in Cocaine Preparations

Sir, The relative long cocaine distribution chain is responsible for cocaine dilution with different cheaper components used as adulterants such as hydroxyzine, lidocaine, procaine, benzocaine, caffeine, boric acid, phenacetin, and levamisole (1). This psychoactive substance modification leads to a weight increase and in consequence to higher profits. Among these adulterants, levamisole gained a lot of attention in recent years, as this drug was reported as especially dangerous to cocaine consumers due to its numerous side effects on human health (agranulocytosis, ANCA-associated vasculitis, retiform purpura, and others) (2,3). Levamisole as an imidazothiazole derivative used to be applied as a veterinary anthelminthic medication but also to treat pediatric nephritic syndrome and rheumatoid arthritis (2). The reason for combining cocaine with levamisole is still unknown. It is believed that levamisole is added directly during cocaine manufacture to potentiate its effects. The prevalence of levamisole-adultered cocaine was investigated recently in North America and was reported to be as high as 88% (4). In this letter, we wanted to report the results of our study concerning the investigation of the levamisole prevalence in cocaine-positive blood samples analyzed in our laboratory and collected during police traffic controls. For these purposes, the LC-MS/MS analytical method used in the routine for the analysis of cocaine and its metabolites was modified to make a qualitative levamisole analysis possible simultaneously. Samples, which contained at least 1 ng/mL benzoylecgonine (cocaine metabolite), were defined as cocaine-positive samples and used in the data evaluation for the purpose of the levamisole prevalence determination. The analysis of 133 cocaine-positive samples in the stretch of 3 months (July–September 2014) revealed that 111 were also positive for levamisole, which equals in a levamisole prevalence of 83%. The LC-MS/MS analytical method applied for the qualitative analysis of levamisole based on the detection of two mass transitions (multiple reaction monitoring mode: 204.9/178.1 for the target and 204.9/123.0 for the qualifier) and was characterized by a limit of detection of 0.14 ng/mL. The presented study revealed that levamisole is present in the majority of cocaine consumed in Lower Saxony. The obtained prevalence was comparable with the data presented by Lynch et al. for cocaine samples in North America. Therefore, this drug has to be considered not only by clinicians in cocaine users with unexpected symptoms such as otherwise unexplained agranulocytosis (5) but also by forensic pathologists in their expertise (2). As the prevalence of levamisole seems to be quite high, and furthermore, different adverse effects of this drug are well known and documented, the application of analytical methods which detect not only cocaine and its metabolites but additionally levamisole can be very useful. Additionally, it has to be considered that the knowledge about adulterants in all illicit drugs and their effect on human health is very important and has a big impact on the correct data interpretation in the forensic/ clinical expertise.

Multiple analyte adduct formation in liquid chromatography-tandem mass spectrometry - Advantages and limitations in the analysis of biologically-related samples

Multiple analyte adduct formation was examined and discussed in the context of reproducible signal detection in liquid chromatography-tandem mass spectrometry applied in the analysis of biologically-related samples. Appropriate infusion solutions were prepared in H2O/methanol (3/97, v/v) with 1 mM sodium acetate and 10 mM acetic acid. An API 4000 QTrap tandem mass spectrometer was used for experiments performed in the negative scan mode (-Q1 MS) and the negative enhanced product ion mode (-EPI). γ‑Hydroxybutyrate and its deuterated form were used as model compounds to highlight both the complexity of adduct formation in popular mobile phases used and the effective signal compensation by the application of isotope-labelled analytes as internal standards.

Adduct Formation-Supported Two-Way Electrospray Ionization Strategy for the Determination of Urinary Creatinine Concentration with LC–MS-MS in Abstinence Control

In this work, an alternative LC-MS-MS strategy for the analysis of urinary creatinine in abstinence control was presented and discussed. The two-way electrospray ionization consisted of two different precursor ions in which fragmentation was used in multiple reaction monitoring experiments. A creatinine adduct ion with sodium and sodium acetate together with the conventional analyte protonation was investigated. Adduct formation and fragmentation was explored by appropriate infusion experiments performed with analyte solutions prepared in different concentrations. The analytical signal was compensated by the application of appropriate isotopically labeled internal standard. The advantages of information carried by precursor ions separated in the mass spectra were pointed out. Sample preparation based solely on sample dilution performed in the final HPLC vial directly. A Luna 5 μm C18 (2) 100 A, 150 mm × 2 mm analytical column together with a mobile phase consisted of H2O/methanol = 3%/97% (v/v) with 10 mmol/L ammonium acetate and 0.1% acetic acid (flow = 0.4 mL/min) were used for the separation performed during a run of 5 min. The linearity was examined in the range of 100-3,000 mg/L. The limit of detection (13 mg/L), limit of quantification (43 mg/L) together with method precision/accuracy, selectivity, stability and matrix effect were tested to be appropriate for forensic applications. The applicability of water as surrogate matrix for method calibration was also examined successfully. The presented strategy was used in the analysis of real samples. No interferences with the creatinine peak eluted at about 1.0 min could be recorded.