Development of an Improved LC–MS/MS Assay for the Quantification of Oximes in KIKO Mouse Plasma

Development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantification of oximes in KIKO mouse plasma.

Monitoring pain management drugs and frequently abused drugs is important for physicians to assess patient compliance. Liquid chromatography-tandem mass spectrometry offers high specificity needed for this purpose. In this report, a novel liquid chromatography-tandem mass spectrometry method for simultaneously monitoring 19 drugs/metabolites in urine was developed and validated. Sample preparation included hydrolysis, dilution and turbulent flow online extraction. Analysis was achieved by reverse phase liquid chromatography and triple-quadruple tandem mass spectrometry. Two fragment ions, one for quantification and the other for assuring identification, were monitored for each analyte. No matrix effect or interference was observed. Lower limits of quantification ranged from 5 to 25 ng/mL. Within the linear range, analytical recovery was between 85.8% and 119.4%. Intra-assay and total coefficient of variations were between 0.2% and 12.7%. This method was compared with mass spectrometry methods offered by two other laboratories using 82 patient samples and 60 spiked urine samples showing 60%-100% agreement. The current method identified more positive samples for all analytes except THCA. The discrepancy in detection rates was primarily due to the different cut-offs used by the other laboratories. A sensitive and specific liquid chromatography-tandem mass spectrometry method was developed for measuring 19 drugs/metabolites in urine important for pain management clinics.

Background Treosulfan and busulfan are alkylating agents administered as the conditioning regimen before hematopoietic stem cell transplantation. High busulfan exposure is associated with drug toxicity and low busulfan exposure with rejection or disease recurrence. There is also an association between treosulfan exposure and early toxicity, such as skin toxicity and mucositis. As both busulfan and treosulfan have narrow therapeutic ranges, therapeutic drug monitoring (TDM) is essential. Since the clinical laboratory tests for both drugs are few, a method that can simultaneously measure both drug concentrations under the same conditions would be helpful in practice. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous measurement of treosulfan and busulfan was developed and validated. Methods For preparing calibrators and quality control materials, commercialized reference standards were used for treosulfan, an active metabolite of treosulfan, S,S-EBDM, and busulfan. Treosulfan-D4 and busulfan-D8 isotopes were used as internal standards (ISs). The analytes were separated from 25 µL of plasma after protein precipitation with acetonitrile containing ISs. After centrifugation, the supernatant was diluted with 3% formic acid and then injected into the liquid chromatography system followed by tandem mass spectrometry. Chromatographic separation was performed using ACQUITY Ι-Class plus ultraperformance liquid chromatography (UPLC) system coupled with a XEVO TQ-XS mass spectrometer operated in positive ion electrospray ionization mode. The analytes and ISs were detected in the multiple reaction monitoring (MRM) mode. The limit of detection (LoD), limit of quantification (LoQ), selectivity, linearity, accuracy, precision, carryover, matrix effect, and sample stability were validated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Results Retention times for treosulfan, S,S-EBDM and IS for both analytes were 0.44 min, 0.43 min, and 0.44 min, respectively. Retention times for busulfan and IS for busulfan were 1.20 min and 1.18 min, respectively. The mass-to-charge (m/z) transitions used for quantification in the MRM mode were as follows: treosulfan 296.2 > 87.1; S,S-EBDM 200.2 > 87.1; and busulfan 264.1 > 55.1. The total run time was 4 min. The LoDs of treosulfan, S,S-EBDM, and busulfan were 0.21, 0.23, and 0.001 µg/mL, respectively. The linear range of the calibration curves of treosulfan, S,S-EBDM, and busulfan spanned concentrations of 3.13–100, 0.63–5.00, and 0.16–5.00 µg/mL, respectively. The total precision of the developed method fulfilled the analytical criteria, except for S,S-EBDM. The LC-MS/MS method was adequately selective and accurate and showed no carry-over. It provided an acceptable matrix effect for treosulfan and busulfan; however, the matrix effect could not be excluded for S,S-EBDM. Busulfan and treosulfan were stable in plasma for 2 h at room temperature. When the samples were stored at 10 °C, busulfan was stable for up to a week and treosulfan was stable for up to 3 days, and both were stable for up to a week when stored frozen at −20 °C. Conclusion We developed the LC-MS/MS method to simultaneously measure treosulfan and busulfan. The method met the validation requirements of the CLSI guidelines and showed good performance. Therefore, this method is expected to be helpful in TDM after treosulfan or busulfan treatment in clinical laboratories.

Plasma amino acid analysis is key to the diagnosis and monitoring of inherited disorders of amino acid synthesis, catabolism and transport. Ion exchange chromatography (IEC) is widely accepted as the gold standard method of analysis, but with the introduction of liquid chromatography tandem mass spectrometry (LC-MS/MS) and liquid chromatography mass spectrometry (LC-MS) methods, this should now be questioned. The analytical performance of three commercially available reagent kits, Waters AccQ Tag™ ULTRA LC-MS, SpOtOn Amino Acids LC-MS/MS and Chromsystems MassChrom® Amino Acid Analysis LC-MS/MS, were evaluated and compared with Biochrom Physiological Amino Acids ion exchange chromatography. Correlation with IEC was assessed by Passing-Bablok regression, concordance correlation coefficients (CCC) and Bland-Altman analysis for 21 common amino acids. Calculation of the total error from imprecision and bias was also used to benchmark performance. The MassChrom® and SpOtOn kits demonstrated acceptable inter-batch imprecision (CV < 10%) and accuracy (mean bias < 10%), whereas the AccQ Tag™ ULTRA kit did not. Good correlation (CCC > 0.95) with Biochrom IEC was demonstrated for 10/21 analytes in both the MassChrom® and SpOtOn kits and 6/21 in the AccQ Tag™ ULTRA kit. The LC-MS assay demonstrated variable analytical performance and correlated poorly with ion exchange chromatography. Both LC-MS/MS assays demonstrated comparable analytical performance and reasonable correlation with ion exchange chromatography. They also confer practical advantages which cannot be realized by ion exchange chromatography, superior specificity and significantly faster analysis time, suggesting that ion exchange chromatography should no longer be described as the gold standard method for plasma amino acid analysis.

Everolimus is an immunosuppressant drug used in solid organ transplantation. Immunoassays and liquid chromatography-mass spectrometry (LC-MS) methods have been used for therapeutic drug monitoring of this drug. In LC-tandem mass spectrometry (MS/MS) methods, both 32-desmethoxyrapamycin and everolimus-d4 have been used as internal standards. To compare 2 internal standards (32-desmethoxyrapamycin and everolimus-d4) for the quantification of everolimus by an LC-MS/MS method. Both 32-desmethoxyrapamycin and everolimus-d4 were introduced in the method validation process with 2 transitions simultaneously monitored for everolimus (975.6 → 908.7 as the quantifier and 975.6 → 926.9 as the qualifier) by an established LC-MS/MS method. The key performance characteristics were lower limit of quantification, accuracy, precision, and comparison with an LC-MS/MS method offered by another laboratory. The lower limit of quantification (LLOQ) was 1.0 ng/mL using either internal standard with an analytical recovery of 98.3%-108.1% across the linear range. The total coefficient of variation for everolimus was 4.3%-7.2% with no significant difference between the 2 internal standards. In comparison with an independent LC-MS/MS method, though everolimus-d4 offered a better slope (0.95 versus 0.83), both internal standards showed acceptable results and had a coefficient of correlation r > 0.98 in the tested concentration range of 1.2-12.7 ng/mL. Although everolimus-d4 offered a more favorable comparison with an independent LC-MS/MS method, both everolimus-d4 and 32-desmethoxyrapamycin had acceptable performance as the internal standards for everolimus quantification by the LC-MS/MS method.

BackgroundAll the enzymatic factors/cofactors involved in nitric oxide (NO) metabolism have been recently found in red blood cells. Increased oxidative stress impairs NO bioavailability and has been described in plasma of coronary artery disease (CAD) patients. The aim of the study was to highlight a potential dysfunction of the metabolic profile of NO in red blood cells and in plasma from CAD patients compared with healthy controls.MethodsWe determined L-arginine/NO pathway by liquid-chromatography tandem mass spectrometry and high performance liquid chromatography methods. The ratio of oxidized and reduced forms of glutathione, as index of oxidative stress, was measured by liquid-chromatography tandem mass spectrometry method. NO synthase expression and activity were evaluated by immunofluorescence staining and ex-vivo experiments of L-[15N2]arginine conversion to L-[15N]citrulline respectively.ResultsIncreased amounts of asymmetric and symmetric dimethylarginines were found both in red blood cells and in plasma of CAD patients in respect to controls. Interestingly NO synthase expression and activity were reduced in CAD red blood cells. In contrast, oxidized/reduced glutathione ratio was increased in CAD and was associated to arginase activity.ConclusionOur study analyzed for the first time the whole metabolic pathway of L-arginine/NO, both in red blood cells and in plasma, highlighting an impairment of NO pathway in erythrocytes from CAD patients, associated with decreased NO synthase expression/activity and increased oxidative stress.

In this study, a solid-phase extraction with liquid chromatography and tandem mass spectrometry method was developed to determine the degree of glycosylation of glycosylation sites and the ratio of free carrier protein to total carrier protein for glycoconjugate vaccines. To remove and enrich the glycosylated peptides, a solid-phase extraction method was developed, optimized, and hyphenated to liquid chromatography-tandem mass spectrometry. The developed solid-phase extraction with liquid chromatography-tandem mass spectrometry method was shown to possess a wide linear dynamic range (0.03-100μg/mL), a high sensitivity (0.03μg/mL for CRM197), good interday and intra-day precision (relative standard deviation of peak area<3.3%), and good recoveries from vaccine matrix (90-105%). Finally, the method was utilized to determine the degree of glycosylation and free carrier protein to total carrier protein ratio for pneumococcal conjugate vaccines and meningococcal vaccines. For quality evaluation of glycoconjugate vaccines, the method could provide more information than the traditional size exclusion chromatography method. Fourteen and twelve reported glycosylation sites for CRM197- and tetanus toxin-based vaccines can be detected, respectively.

A simple, specific, fast and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous analysis of delapril (DEL) and manidipine (MAN) from their combination formulation was developed and validated using fesoterodine as the internal standard (IS). The LC-MS/MS method was carried out on a Luna C8 column (50 × 3.0 mm i.d., 3 µm) with a mobile phase consisting of methanol and 10 mmol L(-1) ammonium acetate (90 : 0, v/v), run at a flow rate of 0.25 mL min(-1). The mass spectrometry method was performed employing positive electrospray ionization operating in multiple reaction monitoring mode, monitoring the transitions of m/z 453.1 → 234.1 for DEL, m/z 611.1 → 167.0 for MAN and m/z 412.2 → 223.0 for IS. The total analysis time was 3 min and the method was linear in the concentration range of 6-1080 ng mL(-1) and 2-360 ng mL(-1) for DEL and MAN, respectively. Parameters investigated for the method validation, such as the specificity, linearity, precision, accuracy and robustness, gave results within the acceptable range. Moreover, the proposed method was successfully applied for the simultaneous determination of DEL and MAN and the results were compared to validated liquid chromatography and capillary electrophoresis methods showing non-significant differences (P = 0.9).

Determination of 6-benzylthioinosine in mouse and human plasma by liquid chromatography–tandem mass spectrometry

A liquid chromatography–tandem mass spectrometry method for the analysis of primary aromatic amines in human urine

Editorial.

A simple, rapid, and sensitive method of liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of vardenafil in rabbit plasma. A simple protein precipitation method with ice-cold acetonitrile was used for plasma extraction. The mass transitions m/z 489⟶151 and m/z 390⟶169 were used to measure vardenafil and tadalafil (internal standard), respectively, with a total assay run time of 6 min. The limit of detection was 0.2 ng/mL. The assay was reproducible with intra-assay and interassay precision ranging 1.17%–9.17% and 1.31%–5.86%, respectively. There was also good intra-assay and interassay accuracy between 89.3%–105.3% and 94%–102% of the expected value, respectively. The linearity range was 0.5–60 ng/mL in rabbit plasma (r2 ≥ 0.99). The measured AUC from 0 to 24 h (AUC0 − 24t) for the test and reference formulations were 174.38 ± 95.91 and 176.45 ± 76.88, respectively. For the test, Cmax and Tmax were 75.36 ± 59.53 ng/mL and 1.42 ± 0.19 h, whereas, for the reference, these were 58.22 ± 36.11 ng/mL and 2.04 ± 0.33 h, respectively. The test formulation achieved a slightly lower AUC0 − 24t value (p > 0.05), higher Cmax values (p > 0.05), faster Tmax (p < 0.05), and almost equal bioavailability compared with the reference formulation.

Teicoplanin is a glycopeptide antibiotic that has become increasingly popular with the spread of methicillin-resistant Staphylococcus aureus. The aim of the study was to develop and validate an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for teicoplanin, and analyze trough teicoplanin concentrations achieved in patients with hematological diseases. The UHPLC-MS/MS method for teicoplanin was developed, validated, and applied in a retrospective analysis of trough plasma teicoplanin concentrations from 305 patients receiving standard dose, and 17 patients receiving therapeutic drug monitoring (TDM)-guided individualized dose. The linear range was 3.9-52.9 mg/L. The imprecision was less than 12%, the limits of detection and quantification were less than 0.13 and 0.72 mg/L, respectively. The sample carry-over and ion suppression were insignificant. In the standard dose group, the median teicoplanin concentrations were 7.5 mg/L (days 3-5) and 8.9 mg/L (on days 6-8); and the proportion of trough levels achieving ≥10 mg/L was 20% (days 3-5) and 38% (days 6-8), respectively. In the TDM-guided individualized dose group, median teicoplanin concentration was higher (16.9 mg/L), and the proportion of trough levels ≥10 mg/L was also higher (77%) when compared with the standard dose group. Based on these results, the present UHPLC-MS/MS method can be considered suitable for routine TDM of teicoplanin. Also, based on the insufficient trough teicoplanin concentrations achieved with standard dose regimen, and the higher trough teicoplanin concentrations achieved with TDM-guided individualized dose regimen, this study highlights the importance of TDM of teicoplanin, especially in high-risk patient groups.

Background Abnormal steroid secretion status has been implicated in a variety of adrenal diseases, such as primary aldosteronism (PA), and adrenocortical carcinoma. However, existing methodological studies to quantify urinary steroids have limitations such as few measurable hormones, complex and time-consuming pre-processing, etc. Therefore, the study aims to establish a robust and fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for multi-steroid profiling in human 24-hour urine, promoting individualized diagnosis. Methods IMCS RT transgenic ß-glucuronidase (activity&amp;gt;200kU/mL) was used to hydrolyze urine samples, with the performance under different hydrolysis durations (0, 0.25, 0.5, 1, 2, 4 hours) evaluated. A 96-well µHLB solid-phase extraction plate was used to extract steroids which were then detected using a Waters Acquity I-Class UPLC system in tandem with a Waters TQ-S triple quadrupole MS/MS system. The performance of the LC-MS/MS method was validated according to the Clinical &amp; Laboratory Standards Institute guidelines. The distribution of 24-hour urinary steroid hormones in sixteen patients with PA and seventeen healthy participants was compared. Results Steroids in 40µL 24-hour urine could be effectively hydrolyzed with 5µL IMCS RT ß-glucuronidase at room temperature for approximately 30 minutes. Positive electrospray ionization using multiple reaction modes was performed for most hormones, except for estrone, estradiol, estriol, aldosterone, and tetrahydroaldosterone. Thirty-five steroids were effectively chromatographic separated using an Acuqity UPLC® HSS C18 column and analyzed in one injection within 25 minutes. The linearity range was 0.012-10ng/mL for 4-androsten-3,11,17-trione, 0.12-100ng/mL for estrone, estradiol, estradiol, 18-oxocortisol, 11-deoxycorticosterone, 18-hydro corticosterone and aldosterone, 1.2-1000ng/mL for androstenedione, dehydroepiandrosterone, 11-oxo etiocholanolone, 6ß-hydroxy cortisol and tetrahydro-11-deoxycorticosterone, 0.024-20ng/mL for 11-ketotestosterone, 0.24-200ng/mL for 18-hydroxycortisol and 11-deoxycortisol, 2.4-2000ng/mL for androsterone and ß-cortolone; 0.006-5ng/mL for 17a-hydroxyprogesterone, 0.06-50ng/mL for dihydrotestosterone, dexamethasone, testosterone and 11-hydroxy progesterone, 0.6-500ng/mL for progesterone, cortisone, cortisol and tetrahydroaldosterone, 6-5000ng/mL for 11ß-hydroxy etiocholanolone, tetrahydrocortisol, 5ß-Pregnane-3a,20a-diol, a-cortolone, urocortisone, allo-3a-tetrahydrocortisol, 11ß-hydroxy androsterone, and 5ß-androstan-3a-ol-17-one. The recovery rate was between 85% and 115%. No obvious matrix effect was observed with internal standard correction. Acceptable intra-assay and inter-assay precision were achieved for all analytes with the total coefficients of variation being &amp;lt;20% at low concentrations, and &amp;lt;15% at medium and high concentrations. No carryover was found. Patients with PA showed significantly different steroid secretion conditions when compared with healthy participants, such as higher urinary median values of aldosterone (16.4 vs. 4.0µg/24h), tertrahydroaldosterone (91.9 vs. 14.4µg/24h), 18-oxocortisol (2.7 vs. 1.3µg/24h), 18-hydroxycortisol (143.6 vs 105.8µg/24h), etc., but lower 11ß-hydroxy etiocholanolone (69.6 vs. 191.7µg/24h) (all p&amp;lt;0.05). Conclusion This LC-MS/MS, combined with a shorter hydrolysis time, enables simultaneous quantification of multiple steroids in a single run, allowing for fast, robust, and accurate applications in disease diagnosis.

A simple, specific, rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of fluticasone propionate (FP) in pharmaceutical formulations was developed and validated using deflazacort as internal standard (IS). The LC-MS/MS method was carried out on a C8 column (50 mm) with a mobile phase consisted of methanol : water (95 : 5, v/v) 100 mM formic acid-50 mM ammonium acetate (90 : 5 : 5, v/v/v). The mass spectrometry method was performed employing positive atmospheric pressure chemical ionization technique, operating in multiple reaction monitoring mode. The chromatographic separation was obtained within 1.5 min and it was linear in the concentration range of 10-1000 ng mL(-1). Moreover, method validation demonstrates acceptable results for the specificity, accuracy, precision and robustness. The proposed method was successfully applied for the quantitative analysis of FP nasal sprays and the results were compared to validated liquid chromatography and capillary electrophoresis methods with photodiode array detectors showing non-significant difference (P > 0.05).