Biphenyl Column Research Articles

Conversion of 5α-Androstane-3α,17β-diol to bis[(4-dimethylamino)phenyl carbamate] derivative for sensitive determination of its rat brain level by LC/ESI-MS/MS.

5α-Androstane-3α,17β-diol (3α,5α-Adiol) is a testosterone-derived neurosteroid and has anxiolytic and analgesic effects via γ-aminobutyric acid type A receptors as with the progesterone-derived neurosteroid, allopregnanolone (AP). Although the psychotropic drug-evoked changes in the brain AP concentration have been intensively studied, those in the brain 3α,5α-Adiol concentration remain poorly understood. One of the causes for this is the limited availability of a validated method for quantifying the brain 3α,5α-Adiol with a sufficient sensitivity and specificity, which is described in this study. To enhance the detectability of 3α,5α-Adiol by electrospray ionization-tandem mass spectrometry (ESI-MS/MS), derivatization with 4-dimethylaminobenzoyl azide was employed. The brain sample was purified by solid-phase extraction and the recovered 3α,5α-Adiol and the deuterated internal standard were derivatized, then measured by liquid chromatography (LC)/ESI-MS/MS with selected reaction monitoring. The derivatized 3α,5α-Adiol, i.e., the bis[(4-dimethylamino)phenyl carbamate] derivative, provided the intense doubly-protonated molecule as the precursor ion, then the specific product ion containing the 3α,5α-Adiol-skeleton by collision-induced dissociation. The detectability of 3α,5α-Adiol was eventually increased 1000-fold by derivatization. Separation of the derivatized 3α,5α-Adiol from its stereoisomers and interfering brain components was achieved using a SunShell Biphenyl column with an isopropyl alcohol-containing mobile phase. A good linearity in the sufficient concentration range, acceptable precision and accuracy, and negligible matrix effect were demonstrated by the validation tests. The animal (rat) study using this method revealed that the brain 3α,5α-Adiol levels were unaffected by the administration of fluoxetine (FLX) and clozapine (CLZ), in contrast to the significant increase of the AP levels. An LC/ESI-MS/MS method capable of quantifying 3α,5α-Adiol in the rat brain using a 20-mg tissue was developed and validated. The brain levels of 3α,5α-Adiol had an entirely different behavior from those of AP due to FLX and CLZ administration.

B-176 Rapid Diagnosis of 21-Hydroxylase Deficiency Using LC-MS/MS for Urinary Unconjugated Steroid Analysis

Abstract Background Urinary steroid profiling through gas chromatography-mass spectrometry (GC-MS) serves as a potent diagnostic tool for detecting abnormal steroid production linked to congenital enzyme deficiencies and steroid-related diseases. This technique involves deconjugating and extracting both conjugated and unconjugated steroids, followed by derivatization and analysis via GC-MS. It provides comprehensive insights into steroid metabolism, aiding in the identification of genetic disorders and conditions with atypical steroid levels. However, the procedure is intricate and time-consuming, particularly the deconjugation of steroids for analysis. The present study aimed to assess the usability of LC-MS/MS assays measuring unconjugated Δ4- and Δ5-steroids in urine for diagnosing steroid abnormalities. Methods The Biotage ISOLUTE SLE+ (Biotage, Sweden) was used for sample preparation, with dichloromethane for elution. LC-MS/MS analysis was performed on an LCMS8060NX (Shimadzu, Kyoto, Japan), using a Shim-Pack Velox Biphenyl column 2.1mm*150mm* 1.8μm (Shimadzu GLC, Tokyo, Japan). The mobile phase consisted of a 0.2mmol/L ammonium fluoride aqueous solution and a 0.2mmol/L ammonium fluoride methanol solution. We targeted 38 steroids, including four steroid medications, and used 19 stable isotope-labeled steroids for quantification. For the measurements, 30 neonatal urine samples were used (IRB Approval Number 20221210), which were suspected of 21-hydroxylase deficiency (21OHD) based on clinical symptoms or screening for 17α-hydroxyprogesterone (17OHP) using Dried Blood Spot. Of these, 18 had been confirmed as 21OHD cases by exceeding the GC-MS diagnostic criterion for Pregnanetriolone (>0.06ng/g Creatinine). Results Blood 21-deoxycortisol (21DOF) is a reliable marker for diagnosing 21OHD, which is generated from 17OHP through an alternative biochemical route. In consideration of that, we measured the urinary concentration of unconjugated 21-deoxycortisone (21DOE, precurcer of Pregnanetriolone), into which 21DOF is putatively converted in the kidney. In cases where 21OHD was excluded by GC-MS diagnosis, 21DOE was undetectable, falling below the sensitivity levels. On the other hand, 21DOE was detected in all samples confirmed 21OHD cases, consistent with the conversion of 21DOF to 21DOE in the kidney. Conclusions The present study suggested the usability of LC-MS/MS assays measuring urinary unconjugated 21DOE for diagnosing 21OHD. This approach provides a faster, less complex method for diagnosis, eliminating the need for the deconjugation process. To establish the value of urinary unconjugated steroids as diagnostic markers for steroid abnormalities, efforts to accumulate further findings are underway using clinical samples.

Characterization of etomidate and emerging analogs in human hair using UHPLC-MS/MS and confirmation in real forensic cases

The regulation of etomidate, a widely used drug for anesthesia induction and short surgical procedures, has led to the emergence of etomidate analogs such as metomidate, propoxate, and isopropoxate. This study introduces and validates a simple, rapid, and cost-effective ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the determination and quantification of etomidate, etomidate acid, metomidate, propoxate, and isopropoxate in human hair. Using a five-minute gradient elution on a Phenomenex Kinetex Biphenyl column, the method achieves low limits of quantification (LOQs), ranging from 5 to 20 pg/mg. Method validation confirms robust linear calibration curves for the target substances in hair samples within the range of LOQs to 1000 pg/mg, with average correlation coefficients (r) all exceeding 0.999. The method also achieves acceptable intra-day and inter-day precision (<15 %) and trueness(bias, −10.9 % to 14.4 %). The matrix effect ranges from 46.9 % to 94.2 %, and the extraction recovery rate ranges from 84.5 % to 105 %. When applied to authentic cases, this method successfully identified 56 positive samples. The concentrations of etomidate, etomidate acid, metomidate, propoxate, and isopropoxate in positive samples ranged from 27 to 1.5 × 105 pg/mg, 21 to 1.5 × 103 pg/mg, 18 to 8.7 × 104 pg/mg, 25 to 1.6 × 104 pg/mg, and 22 to 5.0 × 105 pg/mg, respectively.

Simultaneous multianalyte trace-level quantification of eight genotoxic nitrosamine impurities in valsartan Active Pharmaceutical Ingredient and tablet formulation using UFLC-MS/MS and greenness assessment

Valsartan, a widely prescribed antihypertensive drug, was identified for the presence of carcinogenic nitrosamine impurities in 2018, leading to product recalls worldwide and necessitating sensitive liquid and gas chromatographic methods for trace level detection of nitrosamine impurities. This study developed and validated an ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method for the quantification of eight genotoxic nitrosamines (NDMA, NDEA, NDPA, NEIPA, NDIPA, NMBA, NDBA, and NMPA) in valsartan drug substance and tablet formulations. The method employed a triple quadrupole analyzer, atmospheric pressure chemical ionization (APCI) ionization source and multiple reaction monitoring (MRM) scan mode for the analysis. Chromatographic separation was achieved on a Phenomenex biphenyl column (150 × 4.6 mm, 3 µm) maintained at 55 °C. The mobile phase consisted of a binary gradient of solvent A (0.1 % formic acid in water) and solvent B (0.1 % formic acid in methanol), with a total run time of 20 min for the sensitive and specific detection of the analytes. Method validation demonstrated excellent linearity, recovery, precision, and sensitivity. The greenness of the developed analytical method was critically assessed through comprehensive evaluations using the GAPI, AGREE, and AES metrics and the results obtained are compared with the reported methods in the literature. This novel UFLC-MS/MS method offers a reliable and sensitive approach for routine quality control analysis of nitrosamine impurities in Valsartan drug substance and tablet formulations.

Simultaneous Determination of Tobacco Smoke Exposure and Stress Biomarkers in Saliva Using In-Tube SPME and LC-MS/MS for the Analysis of the Association between Passive Smoking and Stress.

Passive smoking from environmental tobacco smoke not only increases the risk of lung cancer and cardiovascular disease but may also be a stressor triggering neuropsychiatric and other disorders. To prevent these diseases, understanding the relationship between passive smoking and stress is vital. In this study, we developed a simple and sensitive method to simultaneously measure nicotine (Nic) and cotinine (Cot) as tobacco smoke exposure biomarkers, and cortisol (CRT), serotonin (5-HT), melatonin (MEL), dopamine (DA), and oxytocin (OXT) as stress-related biomarkers. These were extracted and concentrated from saliva by in-tube solid-phase microextraction (IT-SPME) using a Supel-Q PLOT capillary as the extraction device, then separated and detected within 6 min by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a Kinetex Biphenyl column (Phenomenex Inc., Torrance, CA, USA). Limits of detection (S/N = 3) for Nic, Cot, CRT, 5-HT, MEL, DA, and OXT were 0.22, 0.12, 0.78, 0.39, 0.45, 1.4, and 3.7 pg mL-1, respectively, with linearity of calibration curves in the range of 0.01-25 ng mL-1 using stable isotope-labeled internal standards. Intra- and inter-day reproducibilities were under 7.9% and 14.6% (n = 5) relative standard deviations, and compound recoveries in spiked saliva samples ranged from 82.1 to 106.6%. In thirty nonsmokers, Nic contents positively correlated with CRT contents (R2 = 0.5264, n = 30), while no significant correlation was found with other biomarkers. The standard deviation of intervals between normal beats as the standard measure of heart rate variability analysis negatively correlated with CRT contents (R2 = 0.5041, n = 30). After passive smoke exposure, Nic levels transiently increased, Cot and CRT levels rose over time, and 5-HT, DA, and OXT levels decreased. These results indicate tobacco smoke exposure acts as a stressor in nonsmokers.

Fast and reliable quantification of aldosterone, cortisol and cortisone via LC-MS/MS to study 11β-hydroxysteroid dehydrogenase activities in primary cell cultures

Cell culture experiments can support characterization of enzymatic activities in healthy and tumorous human tissues. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) enables simultaneous measurement of several steroids from a single sample, facilitating analysis of molecular pathways involved in steroid biosynthesis. We developed a reliable but fast method for quantification of cortisol, cortisone and aldosterone in cell culture supernatant. Validation, including investigation of matrix-matched calibration, was performed for two different cell types. Utility of the method was demonstrated in the study of 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) activity under conditions of glucocorticoid and mineralocorticoid excess in different cell types. Aldosterone, cortisol and cortisone were extracted by liquid-liquid extraction (LLE) with methyl tert-butyl ether from 1 mL of cell culture supernatant. Steroids were separated on a Kinetex biphenyl column (50 ×2.1 mm, 2.6 µm) with gradient elution of water and methanol containing 2 mM ammonium format and analysed in multiple reaction monitoring mode after positive electrospray ionization. Application of the method included cell culture experiments with two different primary cell types, human coronary artery smooth muscle cells (HCSMC) and human coronary artery endothelial cells (EC). Cells were treated with different concentrations of cortisol, aldosterone and mifepristone, a glucocorticoid receptor antagonist and quantitative PCR was performed. The method exhibits high precision (CV ≤ 6 %) and accuracy (deviation from nominal concentration ≤ 6 %) for concentrations above the limit of quantification (LoQ) which is 0.11, 0.56 and 0.69 nmol/L for aldosterone, cortisone and cortisol, respectively. Calibration curves did not differ when prepared in media or solvent. The method enabled us to confirm activity of HSD11B2 and concentration dependent conversion of cortisol to cortisone in HCSMC (median conversion ratio at 140 nM cortisol = 1.46 %). In contrast we did not observe any HSD11B2 activity in EC. Neither addition of high aldosterone, nor addition of 1 µM mifepristone had impact on glucocorticoid concentrations. Quantitative PCR revealed expression of HSD11B1 and HSD11B2 in HCSMC but not in EC. We present a fast and reliable method for quantification of cortisol, cortisone and aldosterone in cell culture supernatants. The method enabled us to study HSD11B2 activity in two different cell types and will support future experiments investigating mechanisms of target organ damage in conditions of glucocorticoid and mineralocorticoid excess.

Substantial benefits of an inert biphenyl column for the analysis of steroids and their phase II metabolites in biological samples.

Steroids can be used as biomarkers in clinical metabolomics and other fields related to human toxicology. This chemical group is known for its complexity, considering its number of isobaric compounds and the wide variety of phases I and II metabolic pathways that parent compounds can undergo. For a successful analysis of steroids in biological samples, liquid chromatography separation must be finely tuned. It is especially challenging for glucuronidated and sulfated steroids derivatives that bear polar heads and can be affected by non-specific adsorption. The benefits of a biphenyl stationary phase chemistry for the selectivity of the separation of steroids and their phase II metabolites and the extent to which nonspecific adsorption phenomena could degrade chromatographic performance were investigated. Replacing a conventional hardware by a passivated hardware allowed to considerably reduce peaks width and asymmetry of sulfated species. The addition of weak ion pairing agents in the mobile phase could also help to reduce non-specific adsorption but are detrimental to mass spectrometry detection. As confirmed by the successful detection of 52 steroids in plasma, the use of a biphenyl stationary phase complemented by a passivated column hardware is of great help for a successful biomedical analysis of steroids and their phase II metabolites.

Validation of LC-MS/MS method for opioid monitoring in Valencia City wastewater: Assessment of synthetic wastewater as potential aid

In this study, a comprehensive and sensitive method for the simultaneous detection of 17 opioids (OPs) and their human metabolites in wastewater using high-performance liquid chromatography coupled to tandem mass spectrometry was validated. The chromatographic separations of opioids were carried out on a Kinetex® Biphenyl column (1.7 μm, 100 Å, 50 × 2.1 mm). A synthetic wastewater approach was used for recovery studies to mimic a contaminant-free matrix. Two solid-phase extraction (SPE) sorbents (hydrophilic-lipophilic balance and mixed mode with the previous phase and a weak cationic exchange) were studied to optimize sample treatment and obtain higher recoveries. The mixed mode was chosen because the recoveries of 17 target analytes at three spiked concentrations (25, 50, and 100 ng mL−1) were > 80 % for 75 % of the analytes in a simulated wastewater. The intra- and inter-day relative standard deviations (RSDs) were between ±1 % and ±20 %. The method limits of quantification ranged from 5 to 25 ng L−1, the only exceptions being heroin (275 ng L−1) and morphine-3β-glucuronide (250 ng L−1). Suppression/enhancement is comparable between the synthetic and the influent wastewater. The analytical method was applied to the OPs analysis in twenty-one influent samples collected from the treatment plants treating the wastewater of Valencia City (Spain). Twelve OPs were detected with total daily concentrations ranging from 1 ng L−1 to 2135 ng L−1. The widespread presence of these compounds in water suggests potential widespread exposure, highlighting the need for increased environmental awareness. Furthermore, the estimated daily intake results raise concerns about opioid use as a potential future health and social issue.

Electromembrane extraction of drugs of abuse and prescription drugs from micropulverized hair.

Hair analysis can provide chronological insights into past drug use for months to years after drug administration. In comparison to analyses from other biological matrices, such as blood and urine, sample pretreatment is often tedious and not environmental friendly. In this study, we present a more environmental friendly approach to hair analysis using micropulverized hair and electromembrane extraction for the efficient extraction of 15 drugs of abuse, prescription drugs, and metabolites from hair. The optimized extraction method, involving micropulverization, demonstrated comparable yields to the standard approach of cutting and overnight incubation. A 15-min extraction method using a commercial electromembrane extraction prototype was developed and validated according to forensic guidelines, using only 10 µl of organic solvent per sample. The final method, employing HPLC-MS-MS with a biphenyl column, exhibited good linearity, precision, and sensitivity. An AgreePrep assessment comparing the environmental impact of our method with the standard routine method, involving overnight incubation and conventional liquid-liquid extraction, was conducted. This is the first time micropulverized hair has been subjected to electromembrane extraction.

On the Surprising Retention Order of Ketamine Analogs Using a Biphenyl Stationary Phase

An unexpected retention order for the ketamine analogs methoxpropamine (MXPr) and methoxisopropamine (MXiPr) was observed when using a biphenyl stationary phase for liquid chromatography-mass spectrometry (LC–MS). For separations of isomeric pairs of drugs (ketamine analogs, tryptamines, fentanyls, and nitazenes), branched-chain compounds are eluted before straight-chain compounds when using acetonitrile or methanol in mobile phases with C18 or biphenyl stationary phases. However, when a biphenyl stationary phase was paired with methanol, the opposite retention order for MXPr and MXiPr occurred, which was not observed when using acetonitrile in the mobile phase. The phenomenon persisted in biphenyl columns from two vendors. This study suggests additional work may be needed to understand the retention mechanisms when using biphenyl stationary phases. It is an example that we can still expect the unexpected when investigating alternative stationary phases of reversed-phase liquid chromatography (RPLC).

A rapid and sensitive LC-MS/MS method for quantifying oxycodone, noroxycodone, oxymorphone and noroxymorphone in human plasma to support pharmacokinetic drug interaction studies of oxycodone.

A sensitive and reliable LC-MS/MS method was developed and validated for the quantification of oxycodone and metabolites in human plasma. The method has a runtime of 6min and a sensitivity of 0.1μg/L for all analytes. Sample preparation consisted of protein precipitation. Separation was performed on a Kinetix biphenyl column (2.1 × 100 mm, 1.7μm), using ammonium formate 5mm in 0.1% aqueous formic acid and methanol LC-MS grade 100% in gradient elution at a flow rate of 0.4ml/min. Detection was performed in multiple reaction monitoring mode using positive electrospray ionization. The method was linear over the calibration range of 0.1-25.0μg/L for oxycodone, noroxycodone and noroxymorphone and 0.1-5.0μg/L for oxymorphone. The method demonstrated good performance in terms of intra- and interday accuracy (86.5-110.3%) and precision (CV 1.7-9.3%). The criteria for the matrix effect were met (CV < 15%) except for noroxymorphone, for which an additional method was applied to compensate for the matrix effect. Whole blood samples were stable for 4h at room temperature. Plasma samples were stable for 24 h at room temperature and 3months at -20°C. Furthermore, the method was successfully applied in a pharmacokinetic drug interaction study of oxycodone and enzalutamide in patients with prostate cancer.

Bioanalytical method optimization for simultaneous quantification of structurally related probe drugs in a phenotyping cocktail using liquid chromatography‐tandem mass spectrometry

AbstractThe physicochemical diversity of the structurally related aromatic probe drugs, used together in a drug cocktail to assess metabolic and transport phenotypes, require optimized analytical procedures for simultaneous quantification. The analytical conditions can greatly influence the analyte selectivity, retention, stability, and ultimately the robustness of the method. The aim of this study was to assess the selectivity of the structurally related ionizable analytes between the commonly used C18 column chemistry and an alternative biphenyl column chemistry as well as the influence of changes in the analytical conditions on method robustness using liquid chromatography‐tandem mass spectrometry. A repeated measure two‐factor analysis of variance with Geisser‐Greenhouse correction was used to determine statistical significance. The results showed that a biphenyl stationary phase in combination with methanol as the organic eluent, could provide improved resolution and analyte selectivity. Changes in analytical conditions caused statistically significant variation in the retention behavior, selectivity, column efficiency, and sensitivity of the analytes of interest The robustness experiment confirmed the importance of controlling analytical conditions to ensure the reproducibility and reliability of the quantitative method.

Diastereomers of Spheroidal Form and Commercially Available Taxifolin Samples

Taxifolin is a natural polyphenol belonging to the class of flavonoids. The structure of this compound is characterized by the presence of two chiral centers. The spheroidal form of taxifolin (TAXs) has emerged as a promising modification due to enhanced solubility, higher safety profile, and long-term release from solid dosage forms. The study’s objective was to assess the diastereomeric content in TAXs and industrially produced samples of taxifolin. Considering the difference in the physico-chemical properties of diastereomers and based on the literature data, we developed a qualitative HPLC method. The chromatograms were recorded using a diode array detector at 290 nm and a mass spectrometer operated in negative ionization mode. Our data suggest that a biphenyl column and gradient elution using 0.1% formic acid in water and 0.2% formic acid in methanol, with the organic phase gradient from 7% to 21% and a flow rate of 0.65 mL/min for 15 min at 60 °C, provides the best conditions for the separation of taxifolin diastereomers. This method was validated for quantitative analysis. We discovered that the cis-isomer was present in all the analyzed samples, with its quantity ranging from 0.8% to 9.5%. TAXs can be considered a sample enriched with diastereomers.

B-205 A Simple Method for Opioids, Synthetic Opioids and PCP Detection in Hair Samples by LC-MS/MS

Abstract Background Hair analysis is useful for monitoring exposure to drugs and is a unique body material for the retrospective detection of drug consumption due to its large detection window, and it is easy to collect, store, and transport. Establishing a LC-MS/MS protocol for opioids determination may be challenging due to similarities among the molecular structures and the MS/MS fragmentation profile. This study aimed to develop and validate a simple, rapid, and sensitive LC-MS/MS method for quantification of 6-acetylcodeine, 6-acetylmorphine, codeine, dihydrocodeine, EDDP, PCP, fentanyl, heroin, hydrocodone, hydromorphone, meperidine, methadone, morphine, oxycodone, oxymorphone and tramadol in hair samples. Methods In this method, 15 mg of hair in a 2.0 mL microtube was decontaminated sequentially with dichloromethane and methanol. The sample was dried, and 0.75 mL of extraction solvent and a labeled internal standards solution were added. The hair sample was pulverized in a FastPrep-24™ 5G from MP-Biomedicals and then sonicated at 60°C for 60 min. The sample was centrifuged and filtered with Captiva filtration plates. Then, 100 µL of supernatant was added to 100 uL of ultrapure water and 5.0 uL was injected directly into the LC-MS/MS system. Chromatographic separation was performed on an Agilent 1290 Infinity II equipped with HALO Biphenyl column, and employing a mobile phase constituted by acetonitrile with 0.1% of formic acid and 5 mM of ammonium formate. The detection was performed with a Sciex 6500+ QTrap mass spectrometer. Results The current method was successfully validated for carryover, linearity, precision, recovery, limits of detection and quantification, and the measurement of uncertainty was calculated. The results were summarized in the Table 1. Conclusion The developed LC-MS/MS method was simple, rapid, and sensitive to detect opioids in hair samples.

B-186 Investigating LC Column Selectivity for 19 Steroids in a Clinical Research Setting

Abstract Background Steroids are a class of structurally related endogenous compounds. Several have the same chemical formula (and hence the same m/z) and must be chromatographically separated for accurate identification and quantitation by LC-MS/MS. Here, six bonded silica solid core or “core-shell” LC columns (see Table 1) were evaluated to determine selectivity and optimum separation conditions for a 19 analyte steroid panel for clinical research. The optimum column for the separation of estrone and estradiol was also established. Methods Analytical reference standards (Cerilliant®, Round Rock, TX) were used to prepare diluted samples for injection. Six different core-shell 2.6 µm, 50 × 2.1 mm LC columns (Kinetex™, Phenomenex, Torrance, CA) were evaluated (see Table 1). Detection employed a 7500 triple quadrupole LC-MS/MS equipped with an ESI source used in positive and negative mode (SCIEX®, Framingham, MA) coupled to an Agilent® 1290 liquid chromatograph (Agilent Technologies, Santa Clara, CA). Results The traditional C18 column worked best for the complete steroid clinical research panel. Estrogens had the best separation on the biphenyl phase, which demonstrated the highest level of selectivity for estrone and estradiol. The phenyl-hexyl column did not sufficiently separate all steroid isomers in the clinical research panel, however it was sufficient to separate estrone and estradiol. Conclusion The proposed HPLC conditions and the core-shell 2.6 μm 50 × 2.1 mm C18 column provided the best separation for 19 steroids with a fast 8 minute run time. For research focused on estrogens, the core-shell biphenyl column yielded the best separation.

B-178 Clinical Routine Ultrasensitive LC-MS/MS Method Development for Analysis of 17β-Estradiol in Human Serum

Abstract Background The increasing clinical demand for the measurement of circulating 17β-Estradiol at very low concentrations consolidates the need of highly sensitive and specific analytical methodologies. These assays are suitable in situations such as antiestrogen therapy monitoring (use of aromatase inhibitor in breast cancer treatment); diagnosis of suspect inherited sex-steroid metabolic disorders; diagnosis of precocious and delayed puberty in female; hormone replacement therapy in postmenopausal women; and estrogen deficiency in men. Time-consuming methods and lack of sensitivity are common in the quantification of 17β-Estradiol in serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The strategy of chemical derivatization is common for analysis of estrogens because it enhances specificity and sensitivity, but this approach becomes laborious in the sample preparation process. For the purpose of solving these analytical challenges, the aim of this work was developing a reliable, sensitive and low-cost LC-MS/MS method without derivatization for clinical routine analysis. Methods Isotope-labeled internal standard 17β-Estradiol-D5 were added to calibrators, quality controls (QC) and serum samples before a liquid-liquid extraction with the organic solvent Diethyl Ether. After reconstitution, the extracts were injected into a Biphenyl column 50 × 2.1 mm, 2,6 µm, chromatographic separated by solvent gradient (Ammonium Fluoride in Water – Acetonitrile) and detected in positive electrospray ionization mode. The analytical method was validated using reference material grade standards and spiked QCs in charcoal stripped pool patient serum at 4 levels by the assessment of linearity, limit of quantification (LoQ), precision inter and intra-assay, accuracy, carryover, method comparison, cross-reactivity and stability (freeze-thawing cycles, bench top, postoperative and stock solution). Results The method has been validated with the analytical measurement range of 2–500 pg/mL. It was successfully optimized to achieve low levels of 17β-Estradiol as proposed with limit of quantitation of 2 pg/mL. Within-run and between-run precision (coefficient of variation) study yielded 1.3%–7.2% and accuracy within 95.7%–112.1%. No carryover, matrix effect and significant cross-reactivity was observed. Estradiol was found to be stable in serum samples under 3 freeze/thawing cycles, 4 h at ambient temperature and 72 h after extraction procedure in auto-injector. The stability of stock solutions was tested in −20 °C freezer and remained stable for 1 month. Method comparison between two reference laboratories (LC-MS/MS method) was performed and the results showed acceptable data for regression statistics (slope (a) = 0.9184; intercept (b) = −0.1576), t-test statistics (bias = 8.5%) and difference plot. Conclusion An ultrasensitive and derivatization-free LC-MS/MS method was developed and it may be beneficial for patients with endocrine disorders associated with estradiol levels.

High-throughput quantification of emerging ''nitazene'' benzimidazole opioid analogs by microextraction and UHPLC-MS/MS.

Benzimidazole opioids, often referred to as nitazenes, represent a subgroup of new psychoactive substances with a recent increase in fatal overdoses in the US and Europe. With a variety of analogs emerging on the illicit drug market, forensic labs are challenged to identify these potent drugs. We here present a simple quantitative approach for the determination of nine nitazene analogs, namely, clonitazene, etodesnitazene, etonitazene, etonitazepyne, flunitazene, isotonitazene, metodesnitazene, metonitazene, and protonitazene in whole blood using liquid-phase microextraction and electromembrane extraction in a 96-well format and liquid chromatography tandem mass spectrometry. Green and efficient sample preparation was accomplished by liquid-phase microextraction in a 96-well format and resulted in high extraction yields for all analytes (> 81%). Here, blood diluted with buffer (1:1, %v) was extracted from a donor compartment across a thin organic liquid membrane and into an aqueous acceptor solution. The acceptor solution was collected and directly injected into the analysis platform. Chromatographic separation was accomplished with a biphenyl column, allowing for baseline separation of the structural isomers isotonitazene and protonitazene before detection by multiple reaction monitoring. The validation was performed according to Scientific Working Group of Forensic Toxicology guidelines. The calibration range was from 0.5 to 50 nM (except for protonitazene and clonitazene from 0.1 nM) with good linearity and limits of detection down to 0.01 nM. An AGREEprep assessment was performed to evaluate sample preparation greenness, with a final score of 0.71. Nitazenes represent a current threat to public health, and analytical methods that cover a wide range of these analogs are limited. The here described method may assist in the detection of nitazenes in whole blood and prevent these substances from being missed in post-mortem investigations.

Quantitative determination of plasma free and total concentrations of antivitamin K drugs using a new sensitive and rapid LC-MS/MS method: Application in hemodialysis patients

Background and aimsVitamin K antagonists (VKAs) are the first-line anticoagulants used in end stage renal disease. This population experiences a significant variability in their International Normalized Ratio (INR) over time. There is a need for methods allowing the study of the pharmacokinetics of free and total concentrations of VKAs to explain INR variability. Materials and methodsWe developed and validated a high-performance liquid chromatography-tandem mass spectrometry method allowing the quantification of warfarin and fluindione free and total plasma concentrations. Chromatographic separation was achieved in a raptor biphenyl column and the spectrometry acquisition was set in multiple reaction monitoring mode after negative electrospray ionization. We then applied it in describing the plasma free and total concentrations of VKAs in samples from 50 hemodialysis patients. ResultsThe developed method is rapid, sensitive and specific. Our cohort results showed a correlation between free and total VKA concentrations. The free VKA concentrations tended to be higher in patients with higher INR. Although VKAs are highly albumin-bound drugs, albumin concentration did not totally explain the high inter-individual total VKA concentrations variability. ConclusionThis opens the door to further studies to understand the factors involved in their variability.

Hair-based rapid UPLC-MS/MS analysis of 36 phencyclidine-type substances in forensic cases

Phencyclidine (PCP) is a frequently abused dissociative agent. It causes confusion, increased tendencies toward violence, and concentration-dependent cytotoxicity after entry into the body. The parent nucleus of phencyclidine-type substances is arylcyclohexylamine, which is easy to modify; therefore, abusers and dealers can readily synthesize substitutes beyond the drug control catalog. An urgent need exists to establish screening methods for phencyclidine-type substances to provide technical support for abuse monitoring. In this study, 20 mg of hair was pulverized in 500 mL of methanol containing 0.5 ng/mL PCP-d5. After ultrasonication, centrifugation, and filtration, the supernatant was analyzed by ultra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) operating in the multiple reaction monitoring mode. Phencyclidine-type substances were separated in 13 min on a biphenyl column using a mobile phase gradient composed of A (water, formic acid 0.1%, acetonitrile 5%, 20 mmol/L ammonium acetate) and B (acetonitrile). The developed and validated method showed good selectivity, sensitivity (limit of detection: 0.25–2 pg/mg and lower limit of quantitation: 0.5–4 pg/mg), linearity (R2 > 0.994), accuracy, and precision (< 20%), and a dilution effect. The method also showed good recovery and acceptable matrix effects for most of the targeted compounds. This analytical approach was successfully applied for the identification and quantification of phencyclidine-type substances in hair from 87 authentic forensic cases. Nine analytes were detected: ketamine (10.3–26211.3 pg/mg), 2-F-2-oxo-PCE (11.5–4034.9 pg/mg), 2-FDCK (14.0–43290.2 pg/mg), 2-BrDCK (10.6–21170.0 pg/mg), nor2-FDCK (10.1–16767.4 pg/mg), tiletamine (10.1–3250.8 pg/mg), O-PCE (43.3–166.1 pg/mg), DCK (10.2–90.4 pg/mg), and norDCK (24.9–103.0 pg/mg).

A UPLC-MS/MS methodological approach for the analysis of 75 phenethylamines and their derivatives in hair

A rapid ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the targeted analysis of 75 phenethylamines and their derivatives from the hair matrix. The monitored classes of phenethylamines included the 2C series, D series, N-benzyl derivatives, mescaline-derived compounds, MDMA analogs, and benzodifurans. Approximately 20 mg of hair was weighed and pulverized with 0.1% formic acid in methanol by cryogenic grinding. After ultrasonication, centrifugation, and filtration, the supernatant was analyzed by LC–MS/MS operating in the scheduled multiple reaction monitoring mode. Phenethylamines and their derivatives were separated in 13 min on a biphenyl column (2.6 µm, 100 Å, 100 × 3.0 mm) using a gradient eluting mobile phase composed of 0.1% formic acid in water and acetonitrile. The developed and validated method showed good selectivity, sensitivity (LOD: 0.5–10 pg/mg and LOQ: 1–20 pg/mg), linearity (R2 > 0.997), accuracy and precision (< 20%), and stability. The method also showed good recovery and acceptable matrix effects for most of the targeted compounds. This analytical approach was successfully applied for the identification and quantification of phenethylamines in hair from authentic forensic cases.