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Kleimaier, Dennis, Victor Schepkin, Cordula Nies, Eric Gottwald, and Lothar R. Schad. "Intracellular Sodium Changes in Cancer Cells Using a Microcavity Array-Based Bioreactor System and Sodium Triple-Quantum MR Signal." Processes 8, no. 10 (October 9, 2020): 1267. http://dx.doi.org/10.3390/pr8101267.
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The sodium triple-quantum (TQ) magnetic resonance (MR) signal created by interactions of sodium ions with macromolecules has been demonstrated to be a valuable biomarker for cell viability. The aim of this study was to monitor a cellular response using the sodium TQ signal during inhibition of Na/K-ATPase in living cancer cells (HepG2). The cells were dynamically investigated after exposure to 1 mM ouabain or K+-free medium for 60 min using an MR-compatible bioreactor system. An improved TQ time proportional phase incrementation (TQTPPI) pulse sequence with almost four times TQ signal-to-noise ratio (SNR) gain allowed for conducting experiments with 12–14 × 106 cells using a 9.4 T MR scanner. During cell intervention experiments, the sodium TQ signal increased to 138.9 ± 4.1% and 183.4 ± 8.9% for 1 mM ouabain (n = 3) and K+-free medium (n = 3), respectively. During reperfusion with normal medium, the sodium TQ signal further increased to 169.2 ± 5.3% for the ouabain experiment, while it recovered to 128.5 ± 6.8% for the K+-free experiment. These sodium TQ signal increases agree with an influx of sodium ions during Na/K-ATPase inhibition and hence a reduced cell viability. The improved TQ signal detection combined with this MR-compatible bioreactor system provides a capability to investigate the cellular response of a variety of cells using the sodium TQ MR signal.
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Jafri, S. H., J. Glass, R. Shi, and H. Kleiner. "Evaluation of thymoquinone (TQ) as an antiproliferative, proapoptotic, and immunomodulatory agent in a non-small cell lung cancer cell (NSCLC) line." Journal of Clinical Oncology 27, no. 15_suppl (May 20, 2009): e14644-e14644. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.e14644.
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e14644 Background: NSCLC is the most common cause of cancer death worldwide. We performed in vitro testing of Thymoquinone (TQ), a derivative of black caraway seed against NSCLC cell line NCI-H460 Methods: Cells were grown in RPMI and were plated at a density of 5,000 cells per well in a 96 well plate and cell growth determined in the presence of 80 and 100μM of TQ dissolved in DMSO with appropriate solvent only as control. Cell proliferation was determined at 24, 48 and 72 hrs intervals using 3-(4,5-dimethyltiazol 2-yl)-2,5-diphenyltetraolium bromide (MTT) assay. Factorial analyses of variance (ANOVA) were used to determine the effect of TQ and control with the time. Student-Newman-Keuls test was used to determine statistical significance with P value <0.05 considered significant. Apoptosis was detected using Annexin V-FITC Aptosis Detection Kit analyzing the effects of TQ at 24 hrs after treatment by flow cytometry. The immunomodulatory effects of TQ were tested using RayBio Human Cytokine Antibody Array C series 200. NCI-H460 cells (50,000 cells per well in duplicate 6 well plates) were grown in serum free RPMI media. 24 hrs after treatment with TQ or DMSO media was collected and analyzed for expression of various cytokines. Results: The MTT assay showed that TQ at 80 and 100 μM significantly inhibited cell growth as compared to control and at 24 hrs for example, 100 μM TQ inhibited cell growth by 78%. Apoptosis occurred rapidly after treatment with TQ with 73.5% of cells being positive for expression of Annexin-V as detected by flow cytometry after 24 hrs of exposure to TQ as compared to 2.6% of controls. The cytokine array revealed that TQ significantly decreased expression of ENA-78(Epithelial neutrophil activating peptide), and GRO (Growth related oncogene).ENA-78 is correlated with vascularity and tumor growth in NSCLC tumor, whereas GRO is associated with neoangiogenesis. Conclusions: TQ is shown to be a powerful anti-proliferative, pro-apoptotic and anti- angiogenic agent in a NSCLC cell line. No significant financial relationships to disclose.
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Soliman, Rabab M., Randa A. Abdel Salam, Basma G. Eid, Ahdab Khayyat, Thikryat Neamatallah, Mostafa K. Mesbah, and Ghada M. Hadad. "Stability study of thymoquinone, carvacrol and thymol using HPLC-UV and LC-ESI-MS." Acta Pharmaceutica 70, no. 3 (September 1, 2020): 325–42. http://dx.doi.org/10.2478/acph-2020-0028.
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AbstractThe aim of this study was to investigate the stability of three major antioxidants of Nigella sativa: thymoquinone (TQ), carvacrol (CR) and thymol (THY), under different stress conditions using HPLC and LC-MS/MS. Forced degradation for each compound was performed under different conditions, including oxidation, hydrolysis, photolysis and thermal decomposition. The results showed that both CR and THY were stable under the studied conditions, whereas TQ was not affected by acidic, basic and oxidative forced conditions but the effect of light and heat was significant. The degradation products of TQ were further investigated and characterized by LC-MS/MS. HPLC-UV method has been fully validated in terms of linearity and range, the limit of detection and quantitation, precision, selectivity, accuracy and robustness. The method was successfully applied to quantitative analysis of the principal antioxidants of Nigella sativa TQ, CR and THY in different phytopharmaceuticals.
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Álvarez-Fernández García, R., M. Corte-Rodríguez, M. Macke, K. L. LeBlanc, Z. Mester, M. Montes-Bayón, and J. Bettmer. "Addressing the presence of biogenic selenium nanoparticles in yeast cells: analytical strategies based on ICP-TQ-MS." Analyst 145, no. 4 (2020): 1457–65. http://dx.doi.org/10.1039/c9an01565e.
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Das, Avijit Kumar, Naoto Hayashi, Yasuhiro Shiraishi, and Takayuki Hirai. "An antimalarial drug, tafenoquine, as a fluorescent receptor for ratiometric detection of hypochlorite." RSC Advances 7, no. 48 (2017): 30453–58. http://dx.doi.org/10.1039/c7ra04867j.
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An antimalarial drug, tafenoquine (TQ), behaves as a fluorescent receptor for ratiometric detection of hypochlorite (OCl−), facilitating rapid, selective, and sensitive detection or imaging of OCl−in solution and living cells.
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Hadad, Ghada M., Randa A. Abdel Salam, Rabab M. Soliman, and Mostafa K. Mesbah. "High-Performance Liquid Chromatography Quantification of Principal Antioxidants in Black seed (Nigella sativa L.) Phytopharmaceuticals." Journal of AOAC INTERNATIONAL 95, no. 4 (July 1, 2012): 1043–47. http://dx.doi.org/10.5740/jaoacint.11-207.
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Abstract A new, simple, sensitive, rapid, and accurate isocratic RP-HPLC method was developed and validated for simultaneous analysis of the principal antioxidants of Nigella sativa, i.e., thymoquinone (TQ), carvacrol (CR), and its isomer thymol (THY), in different phytopharmaceuticals. The mobile phase was water–methanol (40 + 60, v/v) at a flow rate of 1.5 mL/min. Quantification was achieved with UV detection at 254 nm, based on peak area. The method was validated for linearity, accuracy, precision, selectivity, and robustness. The proposed method is stability-indicating for determination of TQ in the presence of its degradants. The LOD and LOQ (μg/mL) were, respectively, 0.006 and 0.021 for TQ, 0.002 and 0.006 for CR, and 0.027 and 0.090 for THY. The mean recoveries measured at three concentrations were higher than 99%, with RSD <2%. This analytical method is suitable for quality control of the marker substances in this widely used natural protective and curative remedy.
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Zhu, Yu-Jun, De-Run Huang, Ye-Yang Fan, Zhen-Hua Zhang, Jie-Zheng Ying, and Jie-Yun Zhuang. "Detection of QTLs for Yield Heterosis in Rice Using a RIL Population and Its Testcross Population." International Journal of Genomics 2016 (2016): 1–9. http://dx.doi.org/10.1155/2016/2587823.
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Analysis of the genetic basis of yield heterosis in rice was conducted by quantitative trait locus mapping using a set of 204 recombinant inbred lines (RILs), its testcross population, and mid-parent heterosis dataset (HMP). A total of 39 QTLs for six yield traits were detected, of which three were detected in all the datasets, ten were common to the RIL and testcross populations, six were common to the testcross and HMP, and 17, 2, and 1 were detected for RILs, testcrosses, and HMP, respectively. When a QTL was detected in both the RIL and testcross populations, the difference between TQ and IR24 and that between Zh9A/TQ and Zh9A/IR24 were always in the same direction, providing the potential to increase the yield of hybrids by increasing the yield of parental lines. Genetic action mode of the 39 QTLs was inferred by comparing their performances in RILs, testcrosses, and HMP. The genetic modes were additive for 17 QTLs, dominance for 12 QTLs, and overdominance for 10 QTLs. These results suggest that dominance and overdominance are the most important contributor to yield heterosis in rice, in which the accumulative effects of yield components play an important role.
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Taleuzzaman, Mohamad, Syed Sarim Imam, and Sadaf Jamal Gilani. "Quantitative Determination of thymoquinone in Nigella Sativa and its nano formulation using validated stability indicating HPTLC densiometric method." International Current Pharmaceutical Journal 6, no. 10 (March 6, 2018): 53–60. http://dx.doi.org/10.3329/icpj.v6i10.35897.
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An accurate and stability indicating high-performance thin layer chromatographic method (HPTLC) was developed and validated for the quantification of thymoquinone (TQ) as per the ICH guidelines. The analysis was carried out on the aluminum plate using n-hexane: ethyl acetate: methanol (7:2:1 v/v/v) as the mobile phase and the densitometric determination was carried out by TLC scanner (CAMAG) at 254 nm. The developed method was validated for different parameters like linearity, precision, recovery, robustness, and stressed stability study. The developed analytical method was found to be linear in the concentration range of 75-500 ng band-1 with regression value closer to unity (r2 = 0.997). The developed system was found to give compact spots for thymoquinone (Rf 0.77) with the limit of detection and limit of quantification (18 and 54 ng band-1) respectively. Further, the study showed accuracy, precision and repeatability were all within the required limits. The stress degradation study of TQ showed well separated degraded peak from the pure thymoquinone. The mean recoveries measured at three concentrations were more than 95% with RSD ≤ 3%. The method has been successfully applied in the analysis and routine quality control of herbal material and formulations containing TQ.Taleuzzaman et al., International Current Pharmaceutical Journal, September 2017, 6(10): 53-60http://www.icpjonline.com/documents/Vol6Issue10/01.pdf
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Feldsine, Philip, Mandeep Kaur, Khyati Shah, Amy Immerman, Markus Jucker, and Andrew Lienau. "AOAC Official MethodSM Matrix Extension Validation Study of Assurance GDSTM for the Detection of Salmonella in Selected Spices." Journal of AOAC INTERNATIONAL 98, no. 4 (July 1, 2015): 930–38. http://dx.doi.org/10.5740/jaoacint.2009_03mod.
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Abstract Assurance GDSTM for Salmonella Tq has been validated according to the AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces for the detection of selected foods and environmental surfaces (Official Method of AnalysisSM 2009.03, Performance Tested MethodSM No. 050602). The method also completed AFNOR validation (following the ISO 16140 standard) compared to the reference method EN ISO 6579. For AFNOR, GDS was given a scope covering all human food, animal feed stuff, and environmental surfaces (Certificate No. TRA02/12-01/09). Results showed that Assurance GDS for Salmonella (GDS) has high sensitivity and is equivalent to the reference culture methods for the detection of motile and non-motile Salmonella. As part of the aforementioned validations, inclusivity and exclusivity studies, stability, and ruggedness studies were also conducted. Assurance GDS has 100% inclusivity and exclusivity among the 100 Salmonella serovars and 35 non-Salmonella organisms analyzed. To add to the scope of the Assurance GDS for Salmonella method, a matrix extension study was conducted, following the AOAC guidelines, to validate the application of the method for selected spices, specifically curry powder, cumin powder, and chili powder, for the detection of Salmonella.
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Krzyzanowska, Justyna, Bogdan Janda, Lukasz Pecio, Anna Stochmal, Wieslaw Oleszek, Anna Czubacka, Marcin Przybys, and Teresa Doroszewska. "Determination of Polyphenols in Mentha longifolia and M. piperita Field-Grown and In Vitro Plant Samples Using UPLC-TQ-MS." Journal of AOAC INTERNATIONAL 94, no. 1 (January 1, 2011): 43–50. http://dx.doi.org/10.1093/jaoac/94.1.43.
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Abstract Nine polyphenols in the aerial parts of Mentha longifolia have been separated by chromatographic techniques. Their structures have been confirmed by HPLC/electrospray ionization-MS/MS. The compounds identified included rosmarinic acid, salvianolic acid L, dedihydro–salvianolic acid, luteolin–glucuronide, luteolin–diglucuronide, luteolin–glucopyranosyl–rhamnopyranoside, and eriodictyol–glucopyranosyl–rhamnopyranoside. The extracts of M. longifolia and M. piperita field plants, in vitro plants, callus tissues, and cell suspension cultures were profiled, and their polyphenol composition was compared in different tissues and quantified using ultra-performance column liquid chromatography (UPLC)/triple-quadrupole-MS in the selected-ion recording detection mode. Determination of desired compounds was based on calibration curves obtained for standards, which were previously isolated from M. longifolia aerial parts. The UPLC profiles revealed considerable differences in the synthesis of secondary metabolites among samples coming from field plants, in vitro plants, callus tissues, and cell suspension cultures. Plant tissues coming from field cultivation (for both M. piperita and M. longifolia) contained several phenolic compounds (flavonoids and phenolic acids), whereas plants from in vitro conditions, callus tissues, and suspension cultures contained only a few of them. Rosmarinic acid dominated in all of these samples. These results show that under in vitro conditions, the metabolism of phenolics undergoes a fundamental change.
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Połyniak, Katarzyna, Przemysław Lisiński, Juliusz Huber, and Leszek Romanowski. "Isokinetic studies for detection of functional disorders in patients with unilateral shoulder impingement syndrome." Journal of Medical Science 83, no. 4 (December 31, 2014): 288–93. http://dx.doi.org/10.20883/medical.e81.
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Introduction. The isokinetic assessment allows objective evaluation of muscle strength in patients considered for surgery in cases of rotator cuff injury. Aim. The goal of this study was to define functional disorders of shoulder joint in patients with rotator cuff injury. Material and methods. The examination was conducted in two groups, ten patients each. One group consisted of subjects with rotator cuff injury while the other was a healthy control group. Isokinetic test was performed with use of Biodex System 4 Pro Device. The following parameters were evaluated: peak torque, peak torque/body weight (peak TQ/BW), total work, average power, range of movement (ROM) and peak torque ratio of external to internal rotators (ERPT/IRPT ratio). Studies of Shoulder Pain and Disability Index (SPADI) scale supplemented clinical evaluation. Results. Examination indicated a significant deficit of muscle strength during external rotation and ROM limitation only on symptomatic side in shoulder impingement group. Alteration between agonistic and antagonistic muscles strength for 2400/s was found. Significant differences between involved and uninvolved shoulders during pain and disability tests were detected. There was no correlation between result of isokinetic and SPADI tests. Conclusions. Patients after injury of rotator cuff present functional disorders that occur mainly during external rotation in isokinetic evaluation.
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Yue, Yaru, Chengdong Chen, Pengkun Liu, Ying Xing, and Xiaoguang Zhou. "Automatic Detection of Short-Term Atrial Fibrillation Segments Based on Frequency Slice Wavelet Transform and Machine Learning Techniques." Sensors 21, no. 16 (August 5, 2021): 5302. http://dx.doi.org/10.3390/s21165302.
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Atrial fibrillation (AF) is the most frequently encountered cardiac arrhythmia and is often associated with other cardiovascular and cerebrovascular diseases, such as ischemic heart disease, chronic heart failure, and stroke. Automatic detection of AF by analyzing electrocardiogram (ECG) signals has an important application value. Using the contaminated and actual ECG signals, it is not enough to only analyze the atrial activity of disappeared P wave and appeared F wave in the TQ segment. Moreover, the best analysis method is to combine nonlinear features analyzing ventricular activity based on the detection of R peak. In this paper, to utilize the information of the P-QRS-T waveform generated by atrial and ventricular activity, frequency slice wavelet transform (FSWT) is adopted to conduct time-frequency analysis on short-term ECG segments from the MIT-BIH Atrial Fibrillation Database. The two-dimensional time-frequency matrices are obtained. Furthermore, an average sliding window is used to convert the two-dimensional time-frequency matrices to the one-dimensional feature vectors, which are classified using five machine learning (ML) techniques. The experimental results show that the classification performance of the Gaussian-kernel support vector machine (GKSVM) based on the Bayesian optimizer is better. The accuracy of the training set and validation set are 100% and 93.4%. The accuracy, sensitivity, and specificity of the test set without training are 98.15%, 96.43%, and 100%, respectively. Compared with previous research results, our proposed FSWT-GKSVM model shows stability and robustness, and it could achieve the purpose of automatic detection of AF.
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M Elalfy, Mahmoud. "Quantitative Detection of Aflatoxin M1, Ochratoxin and Zearalenone in Fresh Raw Milk o f Cow, Buffalo, Sheep and Goat by UPLC XEVO - TQ i n Dakahlia Governorate, Egypt." Open Access Journal of Veterinary Science & Research 4, no. 2 (2019): 1–8. http://dx.doi.org/10.23880/oajvsr-16000181.
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Animals’ derived food as milk is highly nutritive food for human and substitute mother’s milk for children especially after 6 months. To better produce and maintain sa fe milk and milk product, Ultra Performance Liquid Chromatography XEVO - TQ (UPLC) used to monitor mycotoxins as aflatoxin, ochratoxin and zearalenone levels in fresh raw milk of dairy domestic animals. AFL - M1 was detected in all samples in cow, buffalo, she ep and goat milk with different concentration ranged from high, medium and very low with mean 906, 811.1, 1394.86 and 1183.68 ng/L respectively. According worldwide standard for aflatoxin M1 level in milk as EC and according US FDA Limit, all samples of raw milk exceed this limit up to 100ng /l while 50%, 80, 90 and 60% exceed US FDA Limit. Notably, the Egyptian raw milk is free from ochratoxin and zearalenone mycotoxin. Taken collectively , strict strategies should be taken to reduce level of aflatoxin in raw milk between producer and consumer and so reduce risk of adverse effect on health.
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I. Kashchenko, Nina, and Daniil N. Olennikov. "Phenolome of Asian Agrimony Tea (Agrimonia asiatica Juz., Rosaceae): LC-MS Profile, α-Glucosidase Inhibitory Potential and Stability." Foods 9, no. 10 (September 23, 2020): 1348. http://dx.doi.org/10.3390/foods9101348.
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Functional beverages constitute the rapidly increasing part of the functional food section and represent an area with a wide range of products including herbal-based beverages. We carried out screening investigations of the extracts of 85 Rosaceous tea plants. Among the extracts analyzed Agrimonia asiatica herb extract demonstrated the highest inhibitory activity against the enzyme α-glucosidase (20.29 µg/mL). As a result of chromato-mass-spectrometric profiling of A. asiatica herb with high-performance liquid chromatography with photodiode array and electrospray triple quadrupole mass-spectrometric detection (HPLC-PDA-ESI-tQ-MS) 60 compounds were identified, including catechins, ellagitannins, flavones, flavonols, gallotannins, hydroxycinnamates, procyanidins, most for the very first time. The analysis of the seasonal variation of metabolites in A. asiatica herb demonstrated that the phenolic content was highest in summer samples and lower in spring and autumn. HPLC activity-based profiling was utilized to identify compounds of A. asiatica herb with the maximal α-glucosidase inhibitory activity. The most pronounced inhibition of α-glucosidase was observed for agrimoniin, while less significant results of inhibition were revealed for ellagic acid and isoquercitrin. The evaluation of phenolic content in A. asiatica herbal teas with the subsequent determination of α-glucosidase inhibiting potential was discovered. Maximum inhibition of α-glucosidase was observed for hot infusion (75.33 µg/mL) and the minimum for 30 min decoction (159.14 µg/mL). Our study demonstrated that A. asiatica herbal tea is a prospective functional beverage in which dietary intake may help to reduce blood glucose.
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Nanco, Carrol R., Justin L. Poklis, Marzena M. Hiler, Alison B. Breland, Thomas Eissenberg, and Carl E. Wolf. "An Ultra-High-Pressure Liquid Chromatographic Tandem Mass Spectrometry Method for the Analysis of Benzoyl Ester Derivatized Glycols and Glycerol." Journal of Analytical Toxicology 43, no. 9 (August 21, 2019): 720–25. http://dx.doi.org/10.1093/jat/bkz071.
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Abstract Presented is an ultra-high-pressure liquid chromatographic tandem mass spectrometry (UPLC–MS/MS) method developed for the detection of propylene glycol, glycerol, ethylene glycol and diethylene glycol using isotopically labeled standards in urine as part of ongoing studies to evaluate whether urinary propylene glycol and/or vegetable glycerin concentration are indicators of recent use. Propylene glycol and vegetable glycerol are found in many products that are consumed and used including electronic cigarettes (e-cigarettes). E-cigarettes are battery-powered devices used as an alternative to traditional cigarettes. The liquid formulations aerosolized in these devices largely consist of propylene glycol and/or vegetable glycerol. Published reports regarding the ratio of propylene glycol to glycerol content in these formulations ranged from 50:50 to 100 percent of either. For the analysis of urine specimens from both users and non-users of e-cigarettes, calibrators, controls and specimens were derivatized using benzoyl chloride prior to analysis. They were analyzed using a Waters AcQuity Xevo TQ-S Micro UPLC–MS/MS. Chromatographic separation was performed on an AcQuity UPLC BEH C18 1.7 um, 2.1 × 50 mm, column using a 20 mM ammonium formate in water and 20 mM ammonium formate in methanol as the mobile phase. The method was validated using SWGTOX guidelines for linearity, precision and accuracy, stability, carryover and limit of detection. The linear range was determined using a seven-point calibration curve ranging between 0.5 and 100 mcg/mL. The bias for all validation controls was determined to be ±20% of the expected concentrations with CVs of <15%. A total of 124 urine specimens analyzed collected with 50 specimens collected from self-reported non-smokers (cigarettes/e-cigarettes) confirmed cotinine free using the DRI® Cotinine Assay (Thermo Scientific, Waltham, MA) and 74 specimens collected before and after 12 hours self-reported e-cigarettes abstinence e-cigarette users. Propylene glycol and glycerol were determined to have concentration ranges of “none detected” to 1470 and “none detected” to 2950 mcg/mL, respectively.
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Al-Nimer, Marwan S., Sura A. Al-Mahdawi, Namir M. Abdullah, and Akram Al-Mahdawi. "Epileptic Patients are at Risk of Cardiac Arrhythmias: A Novel Approach using QT-nomogram, Tachogram, and Cardiac Restitution Plots." Journal of Neurosciences in Rural Practice 08, no. 01 (January 2017): 007–13. http://dx.doi.org/10.4103/0976-3147.193553.
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ABSTRACT Background: Sudden death is reported in patients who had a history of epilepsy and some authors believed that is due to cardiac arrhythmias. Objectives: This study aimed to predict that the epileptic patients are at risk of serious cardiac arrhythmias by QT-nomogram, tachogram (Lorenz), and cardiac restitution plots. Methods: A total number of 71 healthy subjects (Group I) and 64 newly diagnosed epileptic patients (Group II) were recruited from Al-Yarmouk and Baghdad Teaching hospitals in Baghdad from March 2015 to July 2015 and included in this study. The diagnosis of epilepsy achieved clinically, electroencephalograph record and radio-images including computerized tomography and magnetic image resonance. At the time of entry into the study, an electrocardiography (ECG) was done, and the determinants of each ECG record were calculated. The QT-nomogram, tachogram, and cardiac restitution plots were used to identify the patients at risk of cardiac arrhythmias. Results: Significant prolonged corrected QT corrected (QTc) and JT corrected intervals were observed in female compared with male at age ≥50 years while the TQ interval was significantly prolonged in males of Group II. Eight patients of Group II had a significant pathological prolonged QTc interval compared with undetectable finding in Group I. QT nomogram did not disclose significant findings while the plots of Lorenz and restitution steepness disclose that the patients of Group II were vulnerable to cardiac arrhythmias. Abnormal ECG findings were observed in the age extremities (≤18 years and ≥50 years) in Group II compared with Group I. Conclusion: Utilization of QT-nomogram, restitution steepness, and tachogram plots is useful tools for detection subclinical vulnerable epileptic patient with cardiac arrhythmias.
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Xia, Yong-Gang, Yan Song, Jun Liang, Xin-Dong Guo, Bing-You Yang, and Hai-Xue Kuang. "Quality Analysis of American Ginseng Cultivated in Heilongjiang Using UPLC-ESI−-MRM-MS with Chemometric Methods." Molecules 23, no. 9 (September 19, 2018): 2396. http://dx.doi.org/10.3390/molecules23092396.
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American ginseng (Panax quinquefolium) has long been cultivated in China for the function food and medicine. Here, ultra-high performance liquid chromatography was coupled with electrospray ionization and triple quadrupole mass spectrometry (UPLC-ESI−-TQ-MS) for simultaneous detection of 22 ginsenosides in American ginseng cultivated in Mudanjiang district of Heilongjiang. The extraction conditions also were optimized by a Box Behnken design experiment. The optimized result was 31.8 mL/g as ratio of liquid to raw materials, 20.3 min of extraction time, and 235.0 W of extraction powers. The quantitative MS parameters for these 22 compounds were rapidly optimized by single factor experiments employing UPLC-ESI−-multiple reaction monitoring or multiple ion monitoring (MRM/MIM) scans. Furthermore, the established UPLC-ESI−-MRM-MS method showed good linear relationships (R2 > 0.99), repeatability (RSD < 3.86%), precision (RSD < 2.74%), and recovery (94–104%). This method determined 22 bioactive ginsenosides in different parts of the plant (main roots, hairy roots, rhizomes, leaves, and stems) and growth years (one year to four years) of P. quinquefolium. The highest total content of the 22 analytes was in the hairy roots (1.3 × 105 µg/g) followed by rhizomes (7.1 × 104 µg/g), main roots (6.5 × 104 µg/g), leaves (4.2 × 104 µg/g), and stems (2.4 × 104 µg/g). Finally, chemometric methods, hierarchical clustering analysis (HCA) and partial least squares discrimination analysis (PLS-DA), were successfully used to classify and differentiate American ginseng attributed to different growth years. The proposed UPLC-ESI−-MRM-MS coupled with HCA and PLS-DA methods was elucidated to be a simple and reliable method for quality evaluation of American ginseng.
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Kertys, Martin, Anna Urbanova, Michal Mestanik, Ingrid Tonhajzerova, and Juraj Mokry. "Simultaneous Determination of Total Cortisol and Cortisone in Human Plasma by Liquid Chromatography-Tandem Mass Spectrometry: Method Development, Validation and Preliminary Clinical Application." Current Pharmaceutical Analysis 15, no. 4 (March 19, 2019): 363–70. http://dx.doi.org/10.2174/1573412914666180427094811.
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Background:Cortisol as a major glucocorticosteroid product of the adrenal cortex which has been recognized as a stress biomarker in evaluating stress related disorders for a long time. Plasma concentration of cortisol and its metabolite cortisone are usually changed in physiological and psychological tension, anxiety and depression. In order to study these changes properly, we need a sensitive, accurate and reproducible assay for plasma cortisol and cortisone determination. </P><P> Objective: The aim of this study was to develop a sensitive and robust method for the determination of total cortisol and cortisone in human plasma using mass spectrometry.Methods:A fast, sensitive and selective liquid chromatography-tandem mass spectrometry (LCMS/ MS) method was developed, validated, and then the levels of cortisol and cortisone were determined. Plasma samples cleanup procedure was composed of two steps: the first was a protein precipitation with 1 % formic acid in acetonitrile, and the second was an on-line solid phase extraction (SPE). Afterwards, cortisol and cortisone were separated using a C18 ACQUITY UPLC BEHTM column with a gradient elution. The mobile phase A was 0.1 % formic acid in water, the mobile phase B was 0.1 % methanol. For the detection we used a XEVO TQ-S mass spectrometer operating in the ESI positive mode.Results:The time of analysis was 6.5 minutes and the quantification range was 5-600 ng/mL for cortisol and cortisone, with > 94% recovery for all analytes (cortisol, cortisone and internal standards). The method was validated according to the EMA guideline for bioanalytical method validation.Conclusion:A simple and sensitive LC-MS/MS method was developed and validated for measurement of cortisol and cortisone in human plasma. Our findings indicate that the proposed analytical method is suitable for routine analysis.
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Olennikov, Daniil N., Aina G. Vasilieva, and Nadezhda K. Chirikova. "Fragaria viridis Fruit Metabolites: Variation of LC-MS Profile and Antioxidant Potential during Ripening and Storage." Pharmaceuticals 13, no. 9 (September 22, 2020): 262. http://dx.doi.org/10.3390/ph13090262.
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Fragaria viridis Weston or creamy strawberry is one of the less-known species of the Fragaria genus (Rosaceae family) with a wide distribution in Eurasia and is still in the shadow of more popular relatives F. ananassa (garden strawberry) or F. vesca (wild strawberry). Importantly, there is a lack of scientific knowledge on F. viridis compounds, their stability in the postharvest period, and bioactivity. In this study, metabolites of F. viridis fruits in three ripening stages were characterized with high-performance liquid chromatography with photodiode array and electrospray ionization triple quadrupole mass spectrometric detection (HPLC-PAD-ESI-tQ-MS). In total, 95 compounds of various groups including carbohydrates, organic acids, phenolics, and triterpenes, were identified for the first time. The quantitative content of the compounds varied differently during the ripening progress; some of them increased (anthocyanins, organic acids, and carbohydrates), while others demonstrated a decrease (ellagitannins, flavonols, etc.). The most abundant secondary metabolites of F. viridis fruits were ellagitannins (5.97–7.54 mg/g of fresh weight), with agrimoniin (1.41–2.63 mg/g) and lambertianin C (1.20–1.86 mg/g) as major components. Antioxidant properties estimated by in vitro assays (2,2-diphenyl-1-picrylhydrazyl radical (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) cation radical (ABTS), ferric reducing antioxidant power (FRAP), and oxygen radical absorbance capacity (ORAC)) showed good antioxidant potential in all ripening stages of F. viridis fruits. The pilot human experiment on the effect of F. viridis fruit consumption on the serum total antioxidant capacity confirmed the effectiveness of this kind of strawberry. Postharvest storage of ripe fruits at 4 °C and 20 °C lead to declining content in the majority of compounds particularly ascorbic acid, ellagitannins, and flavonols, with the most significant loss at room temperature storage. These results suggest that F. viridis fruits are a prospective source of numerous metabolites that have potential health benefits.
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Armer, Jane M., and Rebecca L. Allcock. "Urine ethyl glucuronide and ethyl sulphate using liquid chromatography-tandem mass spectrometry in a routine clinical laboratory." Annals of Clinical Biochemistry: International Journal of Laboratory Medicine 54, no. 1 (July 19, 2016): 60–68. http://dx.doi.org/10.1177/0004563216636648.
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Background Detection of alcohol consumption in clients undergoing treatment for alcohol dependence can be difficult. The ethanol metabolites ethyl glucuronide and ethyl sulphate are detectable for longer in urine than either breath ethanol or urine ethanol. Our aim was to develop a liquid chromatography-tandem mass spectrometry method for urine ethyl glucuronide and ethyl sulphate for use in a routine clinical laboratory and define clinical cut-offs in a large population who had not consumed alcohol for at least two weeks. Methods Urine samples were diluted in 0.05% formic acid in HPLC grade water and then directly injected onto a Waters Acquity ultra high performance liquid chromatography coupled to a Waters TQ Detector. Eighty participants were recruited who had not consumed alcohol for at least two weeks to define cut-offs for urine ethyl glucuronide and ethyl sulphate. Samples and alcohol diaries were also collected from 12 alcohol-dependent clients attending a treatment programme. Results The assay was validated with a lower limit of quantitation of 0.20 mg/L for ethyl glucuronide and 0.04 mg/L for ethyl sulphate. Accuracy, precision, linearity and recovery were acceptable. Cut-offs were established for ethyl glucuronide, ethyl sulphate and ethyl sulphate/creatinine ratio (≤0.26 mg/L, ≤0.22 mg/L and ≤0.033 mg/mmol, respectively) in a non-drinking population. The validated cut-offs correctly identified clients in alcohol treatment who were continuing to drink alcohol. Conclusions A simple liquid chromatography-tandem mass spectrometry method for urine ethyl glucuronide and ethyl sulphate has been validated and cut-offs defined using 80 participants who had not consumed alcohol for at least two weeks. This is the largest study to date to define cut-offs for ethyl glucuronide, ethyl sulphate and ethyl sulphate/creatinine ratio.
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Pratt, Mark S., Martijn van Faassen, Noah Remmelts, Rainer Bischoff, and Ido P. Kema. "An antibody-free LC-MS/MS method for the quantification of intact insulin-like growth factors 1 and 2 in human plasma." Analytical and Bioanalytical Chemistry 413, no. 8 (February 10, 2021): 2035–44. http://dx.doi.org/10.1007/s00216-021-03185-y.
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AbstractInsulin-like growth factors 1 and 2 (IGF-1 and IGF-2) are important biomarkers in research and diagnosis of growth disorders. Quantitative analysis is performed using various ligand-binding assays or enzymatic digestion LC-MS/MS methods, whose widespread adoption is hampered by time-consuming sample preparation procedures. We present a simple and fast antibody-free LC-MS/MS method for the quantification of intact IGF-1 and IGF-2 in human plasma. The method requires 50 μL of plasma and uses fully 15N-labelled IGF-1 as internal standard. It features trifluoroethanol (TFE)-based IGF/IGF-binding protein complex dissociation and a two-step selective protein precipitation workflow, using 5% acetic acid in 80/20 acetone/acetonitrile (precipitation 1) and ice-cold ethanol (precipitation 2). Detection of intact IGF-1 and IGF-2 is performed by means of a Waters XEVO TQ-S triple quadrupole mass spectrometer in positive electrospray ionisation (ESI+) mode. Lower limits of quantification were 5.9 ng/mL for IGF-1 and 8.4 ng/mL for IGF-2. Intra-assay imprecision was below 4.5% and inter-assay imprecision was below 5.8% for both analytes. An excellent correlation was found between nominal and measured concentrations of the WHO reference standard for IGF-1. Comparison with the IDS-iSYS IGF-1 immunoassay showed good correlation (R2 > 0.97), although a significant bias was observed with the immunoassay giving substantially higher concentrations. The LC-MS/MS method described here allows for reliable and simultaneous quantification of IGF-1 and IGF-2 in plasma, without the need for enzymatic digestion. The method can be readily implemented in clinical mass spectrometry laboratories and has the potential to be adapted for the analysis of different similarly sized peptide hormones. Graphical abstract
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Sahlan, Muhamad, Seffiani Karwita, Misri Gozan, Heri Hermansyah, Masafumi Yohda, Young Je Yoo, and Diah Kartika Pratami. "Identification and classification of honey's authenticity by attenuated total reflectance Fourier-transform infrared spectroscopy and chemometric method." August-2019 12, no. 8 (August 2019): 1304–10. http://dx.doi.org/10.14202/vetworld.2019.1304-1310.
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Background and Aim: The authentication of honey is important to protect industry and consumers from such adulterated honey. However, until now, there has been no guarantee of honey's authenticity, especially in Indonesia. The classification of honey is based on the bee species (spp.) that produces it. The study used honey from sting bee Apis spp. and stingless bee Tetragonula spp. based on the fact that the content off honey produced between them has differences. Authenticating honey with currently available rapid detection methods, such as 13C nuclear magnetic resonance analysis, is costly. This study aimed to develop an inexpensive, fast, precise, and accurate classification method for authenticating honey. Materials and Methods: In this study, we use attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy with wavelengths ranging between 550 and 4000 cm-1 as an alternative analysis method, which is relatively less expensive. The spectra of authentic and fake honey samples were obtained using ATR-FTIR and plotted using chemometric discriminant analysis. The authentic honey samples were acquired from a local Indonesian breeder of honey bees, while the fake honey samples were made from a mixture of water, sugar, sodium bicarbonate, and authentic honey. Data were collected using Thermo Scientific's OMNIC FTIR software and processed using Thermo Scientific's TQ Analyst software. Results: Our method effectively classified the honey as authentic or fraudulent based on the FTIR spectra. To authenticate the honey, we formed two classes: Real honey and fake honey. The wavelengths that can best differentiate between these two classes correspond to four regions: 1600-1700 cm-1; 1175-1540 cm-1; 940-1175 cm-1; and 700-940 cm-1. Similarly, for classification purpose, we formed two classes: Apis spp. and Tetragonula spp. The wavelength region that can best classify the samples as belonging to the Apis spp. or Tetragonula spp. class is explicitly within the range of 1600-1700 cm-1. Conclusion: This study successfully demonstrated a method to rapidly and accurately classify and authenticate honey. ATR-FTIR is a useful tool to test the authenticity of honey. Keywords: Apis spp., attenuated total reflectance Fourier transform infrared, discriminant, spectrum, Tetragonula spp.
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Pompei. "Feasibility of Lactobacilli Concentration Detection in Beer by Automated Impedance Technique." Technical Quarterly, 2012. http://dx.doi.org/10.1094/tq-49-1-0315-01.
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"Pre-enrichment for PCR Detection of Beer-Spoiling Yeast – Possibilities and Pitfalls." Technical Quarterly 57, no. 3 (2020). http://dx.doi.org/10.1094/tq-57-3-1015-01.
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"Open-Source PCR and Agar-Based Methods for Cost-Effective Detection of Diastatic Yeast." Technical Quarterly 58, no. 2 (2021). http://dx.doi.org/10.1094/tq-58-2-0611-01.
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Dal Bello, Federica, Michael Zorzi, Riccardo Aigotti, Davide Medica, Vito Fanelli, Vincenzo Cantaluppi, Eleonora Amante, Viviana Teresa Orlandi, and Claudio Medana. "Targeted and untargeted quantification of quorum sensing signalling molecules in bacterial cultures and biological samples via HPLC-TQ MS techniques." Analytical and Bioanalytical Chemistry, November 18, 2020. http://dx.doi.org/10.1007/s00216-020-03040-6.
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AbstractQuorum sensing (QS) is the ability of some bacteria to detect and to respond to population density through signalling molecules. QS molecules are involved in motility and cell aggregation mechanisms in diseases such as sepsis. Few biomarkers are currently available to diagnose sepsis, especially in high-risk conditions. The aim of this study was the development of new analytical methods based on liquid chromatography-mass spectrometry for the detection and quantification of QS signalling molecules, including N-acyl homoserine lactones (AHL) and hydroxyquinolones (HQ), in biofluids. Biological samples used in the study were Pseudomonas aeruginosa bacterial cultures and plasma from patients with sepsis. We developed two MS analytical methods, based on neutral loss (NL) and product ion (PI) experiments, to identify and characterize unknown AHL and HQ molecules. We then established a multiple-reaction-monitoring (MRM) method to quantify specific QS compounds. We validated the HPLC-MS-based approaches (MRM-NL-PI), and data were in accord with the validation guidelines. With the NL and PI MS-based methods, we identified and characterized 3 and 13 unknown AHL and HQ compounds, respectively, in biological samples. One of the newly found AHL molecules was C12-AHL, first quantified in Pseudomonas aeruginosa bacterial cultures. The MRM quantitation of analytes in plasma from patients with sepsis confirmed the analytical ability of MRM for the quantification of virulence factors during sepsis.
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Castro-Cerritos, Karla Viridiana, Julio Cesar Torres-Elguera, Jaqueline Capataz-Tafur, Erick Adrian Juarez-Arellano, and Adolfo Lopez-Torres. "Microwave Assisted DNA Hydrolysis for Global Methylation Analysis by Gas Chromatography/Tandem Mass Spectrometry." Journal of the Mexican Chemical Society 62, no. 2 (June 6, 2018). http://dx.doi.org/10.29356/jmcs.v62i2.398.
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<div><p class="Abstract">The analysis of the global DNA methylation, calculated as the percentage of 5-methylcytosine (5mC) over the total sum of cytosines, is a well stablished biomarker for monitoring large scale epigenetic events in organisms. DNA purification, hydrolysis, separation and detection methods are critical steps to determine this biomarker. In the present work is proposed a robust procedure for DNA acid-hydrolysis assisted by microwave that provides identical DNA methylation patterns that enzymatic hydrolysis and better release of 5mC than acid classic method. The quantification was performed using a gas chromatographer coupled to a mass spectrometer with triple quadrupole as mass analyzer (GC-TQ-MS/MS) using multiple reaction monitoring (MRM) mode for the trimethylsilyl-derivates of nucleobases; following the transitions of 254→238, 240→170 and 254→238, 254→184 (m/z) for C and 5mC respectively, was achieved a limit of detection of 0.46 fmol for C and 0.41 fmol for 5mC. The proposed procedure is capable of determine 0.004% of 5mC in 50 ng of DNA in a chromatographic time of 10 minutes, being a good alternative to LC-MS/MS analysis.</p></div>
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Chen, Peilin, Chunyu Huang, Qing Sun, Huixian Zhong, Feng Xiong, Su Liu, Zhihong Yao, et al. "Granulocyte-Macrophage Colony Stimulating Factor in Single Blastocyst Conditioned Medium as a Biomarker for Predicting Implantation Outcome of Embryo." Frontiers in Immunology 12 (June 30, 2021). http://dx.doi.org/10.3389/fimmu.2021.679839.
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BackgroundIt is highly desirable to develop new strategies based on secretomics to more accurately selection of embryos with the highest developmental potential for transfer. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been reported to promote embryo development and pregnancy establishment. However, the predictive value of GM-CSF in single blastocyst selection remains unclear. This study is to determine the concentration of GM-CSF in human single-blastocyst conditioned medium (SBCM) and to evaluate its association with embryo quality and pregnancy outcome.MethodsThe patients with ≤38 years of age receiving the first cycle of assisted reproductive therapy were included in this study. The patients who had <4 top-quality embryos formed by the fertilized two pronuclear zygotes on day 3 were excluded. A total of 126 SBCM samples (SBCMs) were included, of which blastocysts from 77 SBCMs were later transferred in subsequent frozen-thawed embryo transfer. The concentrations of GM-CSF were detected by single-molecule array (SIMOA) and analyzed for their possible association with embryo quality and pregnancy outcomes. The top-quality embryo (TQ), positive HCG (HP), clinical pregnancy (CP), and ongoing pregnancy (OP) rates were determined and compared between groups divided based on GM-CSF concentrations.ResultsThe detection rate of GM-CSF was found to be 50% in all SBCMs. There were significant differences in TQ rate, HP rate, CP rate and OP rate among high concentration group, medium concentration group and low concentration group. Both GM-CSF alone or GM-CSF combined with the morphological score (MS) had a greater AUC of ROC curve than that of MS alone to predict the pregnancy outcome, and GM-CSF combined with MS had the highest AUC.ConclusionsThe concentration of GM-CSF in SBCM was detected at fg/ml levels, which was associated with embryo quality and pregnancy outcome. Collectively, GM-CSF may be used as a biomarker for prediction of pregnancy outcome and selection of embryos with high developmental potential for transfer in assisted reproductive technology (ART).
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Gallagher, Tara, Stefan Riedel, Joseph Kapcia, Lindsay J. Caverly, Lisa Carmody, Linda M. Kalikin, Junnan Lu, et al. "LC-MS detection of antibiotic agents in sputum from persons with cystic fibrosis." Antimicrobial Agents and Chemotherapy, November 2, 2020. http://dx.doi.org/10.1128/aac.00927-20.
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Antibiotic therapy is expected to impact host microbial communities considerably, yet many studies focused on microbiome and health are often confounded by limited information about antibiotic exposure. Given that antibiotics have diverse pharmacokinetic and antimicrobial properties, investigating the type and concentration of these agents in specific host specimens would provide much needed insight into their impact on the microbes therein. Here, we developed liquid chromatography mass spectrometry (LC-MS) methods to detect 18 antibiotic agents in sputum from persons with cystic fibrosis. Antibiotic spike-in control samples were used to compare three liquid extraction methods on the Waters Acquity Quattro Premier XE. Extraction with dithiothreitol captured the most antibiotics and was used to detect antibiotics in sputum samples from 11 people with cystic fibrosis, with results being compared to the individuals' self-reported antibiotic use. For the sputum samples, two LC-MS assays were used; the Quattro Premier detected nanomolar or micromolar concentrations of 16 antibiotics, whereas the Xevo TQ-XS detected all 18 antibiotics, most at sub-nanomolar levels. In 45% of tested sputum samples (71/158), at least one antibiotic that was not reported by the subject was detected by both LC-MS methods, a discordance largely explained by the thrice weekly administration and long half-life of azithromycin. For ∼37% of samples, antibiotics reported as taken by the individual were not detected by either instrument. Our results provide an approach for detecting a variety of antibiotics at the site of infection, thereby providing a means to include antibiotic usage data into microbiome studies.
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Molina, I., F. Salvador, A. Sánchez-Montalvá, M. A. Artaza, R. Moreno, L. Perin, A. Esquisabel, L. Pinto, and J. L. Pedraz. "Pharmacokinetics of Benznidazole in Healthy Volunteers and Implications in Future Clinical Trials." Antimicrobial Agents and Chemotherapy 61, no. 4 (February 6, 2017). http://dx.doi.org/10.1128/aac.01912-16.
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ABSTRACT Despite its toxicity and low efficacy in the chronic phase, benznidazole is the drug of choice in Chagas disease. Scarce information about pharmacokinetics and pharmacodynamics of benznidazole has been published. We performed a phase I, open-label, nonrandomized pharmacokinetic study of benznidazole (Abarax) conducted with 8 healthy adult volunteers at the Infectious Diseases Department of the Vall d'Hebron University Hospital (Barcelona, Spain). The separation and detection of benznidazole were performed on a Waters Acquity ultraperformance liquid chromatography system (UPLC) coupled with a Waters Xevo TQ MS triple quadrupole mass spectrometer. The pharmacokinetic parameters were calculated based on a noncompartmental body model using Phoenix WinNonlin version 6.3 software. Furthermore, computational simulations were calculated for the multiple-dose administration at two dose regimens: 100 mg of benznidazole administered every 8 h and 150 mg of benznidazole administered every 12 h. After benznidazole administration, the median area under the concentration-time curve from time zero to time t (AUC0–t ) and extrapolated to infinity (AUC0–∞) were about 46.4 μg · h/ml and 48.4 μg · h/ml, respectively. Plasma benznidazole concentrations peaked at 3.5 h, with maximal concentrations of 2.2 μg/ml, and benznidazole exhibited a terminal half-life of 12.1 h. The median maximum concentration (C max) of benznidazole was lower in men than in women (1.6 versus 2.9 μg/ml), and median volume of distribution (V) as a function of bioavailability (F) was higher in men than in women (125.9 versus 88.6 liters). In conclusion, dose regimens (150 mg/12 h or 100 mg/8 h) reached a steady-state range concentration above of the minimum experimental therapeutic dose. Sex differences in the benznidazole pharmacokinetics were observed; mainly, men had lower C max and higher V/F than women.
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Grant, Kirsten M., Craig Livie, Karen Smith, Chui Ha Leung, and Susan Johnston. "A rapid liquid chromatography-tandem mass spectrometry method for the analysis of urinary 5-hydroxyindoleacetic acid (5-HIAA) that incorporates a 13C-labelled internal standard." Annals of Clinical Biochemistry: International Journal of Laboratory Medicine, August 14, 2021, 000456322110380. http://dx.doi.org/10.1177/00045632211038021.
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Background Urinary 5-hydroxyindoleacetic acid (5-HIAA) is a first-line investigation for gastrointestinal neuroendocrine tumours that secrete serotonin. It also has clinical utility for monitoring disease progression and therapeutic response. Aim To develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for urinary 5-hydroxyindoleacetic acid that incorporates a supported liquid extraction and 13C-labelled internal standard. Methods Samples were diluted in ammonium acetate containing a 13C-labelled internal standard (5-hydroxyindole-3a,4,5,6,7,7a-13C6-3-acetic acid). Supported liquid extraction was performed followed by chromatographic separation using the 2.1 × 30 mm CORTECS® UPLC® T3 column. Mass spectrometry detection (Waters Xevo TQ-XS) was performed in electrospray positive mode using the transitions 192.3 > 146.4 m/z (quantifier) and 192.3 > 118.4 m/z (qualifier) for 5-hydroxyindoleacetic acid and 198.2 > 152.4 m/z for 13C-5-HIAA. Results A well-defined 5-hydroxyindoleacetic acid peak was observed at 0.8 min with a run time of 2.4 min. The assay was linear (r2 > 0.99) to 382 µmol/L, with a lower limit of quantification of 5.3 µmol/L (CV <15%). Analysis of 29 external quality assurance samples showed good agreement between our method and the UKNEQAS method mean (4.7% positive bias). The intra- and inter-assay precision was within acceptable limits, and the assay was stable up to 96 h postextraction with minimal carryover. Conclusion We have developed a robust LC-MS/MS method with semi-automated extraction that offers an improved run time and performance over the existing, labour-intensive, HPLC method. The method was quick, precise, showed good agreement with UKNEQAS external quality assurance material and is in routine service for clinical samples.