Development and validation of a method for quantification of human insulin and its synthetic analogues in plasma and post-mortem sera by LC-MS/HRMS.

Analysis of human insulin and its synthetic analogues is increasingly requested for clinical monitoring, for anti-doping purposes, but also for forensic cases. Indeed, insulin analogues may be abused for suicide or homicide - whence their forensic interest. Collection and storage conditions, as well as the phenomenon of degradation make post-mortem serum samples analytically challenging and consequently, the rate of exogenous insulin administration as cause of death is undoubtedly underestimated. However, with recent technological advances and the development of new extraction techniques particularly for anti-doping analyses, detection of insulins in post-mortem samples seems to be achievable. This study describes the first validated quantitative method for analysis human insulin and its six analogues (lispro, aspart, glulisine, glargine, detemir and degludec) in plasma and post-mortem sera. Various extraction processes, namely precipitation + solid phase extraction (SPE), filtration + SPE, precipitation + SPE + immunopurification, and filtration + immunopurification, were assessed to evaluate the lowest limit of detection for all target analogues. The selected sample preparation consists of filtration step followed by immunopurification extraction with an anti-body precoated ELISA plate for plasma. For post-mortem sera, the first step of precipitation was added to remove matrix interferences. The extracts were analyzed by ultra-high-performance liquid chromatography-high resolution mass spectrometry (LC-HRMS), interfaced by electrospray (ESI). The method was validated with respect linearity, precision, accuracy, recovery, matrix effect, dilution and carryover. The limit of quantification (LOQ) in plasma was 0.5 ng/mL for human insulin and rapid-acting insulins, 1.0 ng/mL for glargine, 2.5 ng/mL for degludec and 10 ng/mL for detemir. Two types of post-mortem sera were studied based on the post-mortem interval (PMI): inferior or superior to 48 h. The obtained LOQ were the same for each analogue, independent from the PMI: 1.0 ng/mL for human insulin and rapid-acting insulins, 1.0 ng/mL for glargine, 2.5 ng/mL for degludec and 10 ng/mL for detemir. At the LOQ level, for all insulins and all samples, accuracy was between 70 and 130% and precision inferior to 30%. The validated method was applied to five subjects participating in therapeutic monitoring of insulin and to seven post-mortem cases.

Hydrophilic interaction liquid chromatography and accurate mass measurement for quantification and confirmation of morphine, codeine and their glucuronide conjugates in human urine.

A hydrophilic interaction liquid chromatography-time-of-flight mass spectrometry (HILIC-TOFMS) method for the quantification and confirmation of morphine (M), codeine (C), morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G) and codeine-6-glucuronide (C6G) is presented. The method was validated in terms of specificity, selectivity, extraction recovery, accuracy, repeatability, linearity and matrix effect. After a straightforward sample preparation by solid phase extraction (SPE) the compounds were analyzed directly without the need for hydrolysis, solvent transfer, evaporation or reconstitution. The HILIC technique provided good chromatographic separation which was critical for isomers M3G and M6G. The analytes were detected after electrospray ionization (ESI) in positive mode with mass accuracies below 2 mDa using a 5-mDa window. A measurement range of 50-5000 ng/ml was applied for calibration using deuterated analogs as internal standards. The precision of the method was 5.7% and 10.2% (RSD) within and between days, respectively. The applicability of the method was demonstrated with authentic urine samples known to contain codeine and/or morphine and their intact glucuronide conjugates. Identification of the analytes was based on in-source collision induced dissociation (ISCID), applying three diagnostic ions with accurate mass.

Liquid chromatography/atmospheric pressure ionization-mass spectrometry in drug metabolism studies.

The study of the metabolic fate of drugs is an essential and important part of the drug development process. The analysis of metabolites is a challenging task and several different analytical methods have been used in these studies. However, after the introduction of the atmospheric pressure ionization (API) technique, electrospray and atmospheric pressure chemical ionization, liquid chromatography/mass spectrometry (LC/MS) has become an important and widely used method in the analysis of metabolites owing to its superior specificity, sensitivity and efficiency. In this paper the feasibility of LC/API-MS techniques in the identification, structure characterization and quantitation of drug metabolites is reviewed. Sample preparation, LC techniques, isotope labeling, suitability of different MS techniques, such as tandem mass spectrometry, and high-resolution MS in drug metabolite analysis, are summarized and discussed. Automation of data acquisition and interpretation, special techniques and possible future trends are also the topics of the review.

Analysis of free and esterified sterols in fats and oils by flash chromatography, gas chromatography and electrospray tandem mass spectrometry.

A method for the analysis of free and esterified sterols has been developed. Fat or oil samples were separated on solid-phase extraction silica gel columns into a sterol ester fraction, a fraction of triacylglycerols, and a free sterol fraction containing partial acylglycerols and residual triacylglycerols. Sterol esters and acylglycerols of the free sterol fraction were transesterified to methyl esters. The fatty acid methyl esters from sterol ester fraction and the free sterols from sterol ester fraction and free sterol fraction were determined by GLC. Precursor ion electrospray MS-MS of sterol fragment ions of sterol ester fractions were recorded and used for determination of sterol ester proportions in butterfat and vegetable oil samples.

Electrospray and atmospheric pressure chemical ionization tandem mass spectrometric behavior of eight anabolic steroid glucuronides.

Mass spectrometric and tandem mass spectrometric behavior of eight anabolic steroid glucuronides were examined using electrospray (ESI) and atmospheric pressure chemical ionization (APCI) in negative and positive ion mode. The objective was to elucidate the most suitable ionization method to produce intense structure specific product ions and to examine the possibilities of distinguishing between isomeric steroid glucuronides. The analytes were glucuronide conjugates of testosterone (TG), epitestosterone (ETG), nandrolone (NG), androsterone (AG), 5alpha-estran-3alpha-ol-17-one (5alpha-NG), 5beta-estran-3alpha-ol-17-one (5beta-NG), 17alpha-methyl-5alpha-androstane-3alpha,17beta-diol (5alpha-MTG), and 17alpha-methyl-5beta-androstane-3alpha,17beta-diol (5beta-MTG), the last four being new compounds synthesized with enzyme-assisted method in our laboratory. High proton affinity of the 4-ene-3-one system in the steroid structure favored the formation of protonated molecule [M + H]+ in positive ion mode mass spectrometry (MS), whereas the steroid glucuronides with lower proton affinities were detected mainly as ammonium adducts [M + NH4]+. The only ion produced in negative ion mode mass spectrometry was a very intense and stable deprotonated molecule [M - H]- . Positive ion ESI and APCI MS/MS spectra showed abundant and structure specific product ions [M + H - Glu]+, [M + H - Glu - H2O]+, and [M + H - Glu - 2H2O]+ of protonated molecules and corresponding ions of the ammonium adduct ions. The ratio of the relative abundances of these ions and the stability of the precursor ion provided distinction of 5alpha-NG and 5beta-NG isomers and TG and ETG isomers. Corresponding diagnostic ions were only minor peaks in negative ion MS/MS spectra. It was shown that positive ion ESI MS/MS is the most promising method for further development of LC-MS methods for anabolic steroid glucuronides.