A rapid and robust liquid chromatography/tandem mass spectrometry method for simultaneous analysis of anti-tuberculosis drugs—Ethambutol and pyrazinamide in human plasma

Ethambutol and pyrazinamide are two first-line anti-tuberculosis drugs. Though they are normally combined for the treatment, their highly different polarity complicates simultaneous liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of these two drugs in human plasma with decent peak shape and retention. Here we report a rapid and robust LC/MS/MS method for the simultaneous determination of these two drugs in human plasma. Human plasma samples, together with the isotopically labeled internal standards were extracted using protein precipitation, and then separated on a Chromolith SpeedROD RP-18e column and detected with mass spectrometry. The mobile phase is 0.1% trifluoroacetic acid in water and 0.1% trifluoroacetic acid in methanol. Addition of trifluoroacetic acid in the mobile phases was found to be able to improve peak shape as well as to increase the retention of ethambutol, thus being able to analyze these two drugs at the same time with both drugs having decent peak shape and enough retention on a C₁₈ column. An atmospheric pressure chemical ionization interface was chosen to reduce ion suppression from sample matrix components and provide high sensitivity. The standard curve range was 10.0–5000 ng/mL for ethambutol and 50.0–25,000 ng/mL for pyrazinamide using a plasma sample volume of 50.0 μL. This method has a very short run time of 3.8 min. The method has been fully validated, and <15% relative standard deviation was obtained for both analytes.     

Simultaneous HPLC–UV determination of rhein and aceclofenac in human plasma

Diacerein and aceclofenac are prescribed for reducing the symptoms associated with osteoarthritis. We present a simple HPLC method with UV detection for simultaneous determination of rhein (the immediate metabolite of diacerein) and aceclofenac from human plasma samples. Sample preparation was accomplished through liquid–liquid extraction with ethyl acetate and chromatographic separation was performed on a reversed-phase ODS column. Mobile phase consisted of a mixture of acetate buffer and acetonitrile run under gradient at flow rate of 1.0 ml/min. Wavelength was set at 258 nm. The method was validated for linearity, accuracy, precision and stability. The calibration was linear over the range of 0.1–7.0 μg/ml for rhein and 0.5–20 μg/ml for aceclofenac using 500 μl plasma samples. Extraction recoveries were 85% for rhein and 70% for aceclofenac. The method can easily be adopted for high-throughput clinical and pharmacokinetic studies of above two-drug fixed dose combination formulations.     

Simultaneous quantitative determination of olmesartan and hydrochlorothiazide in human plasma and urine by liquid chromatography coupled to tandem mass spectrometry

A specific, sensitive and rapid method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) was developed for the simultaneous determination of olmesartan (OLM) and hydrochlorothiazide (HCTZ) in human plasma and urine. Solid-phase extraction (SPE) was used to isolate the analytes from biological matrices followed by injection of the extracts onto a C₁₈ column with isocratic elution. Detection was carried out on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode using negative electrospray ionization (ESI). The method was validated over the concentration range of 1.00–1000 ng/mL and 5.00–5000 ng/mL for OLM in human plasma and urine as well as 0.500–200 ng/mL and 25.0–25,000 ng/mL for HCTZ in human plasma and urine, respectively. Inter- and intra-run precision of OLM and HCTZ were less than 15% and the accuracy was within 85–115% for both plasma and urine. The average extraction recoveries were 96.6% and 92.7% for OLM, and 87.2% and 72.1% for HCTZ in human plasma and urine, respectively. The linearity, recovery, matrix effect and stability were validated for OLM/HCTZ in human plasma and urine.     

Stability evaluation and sensitive determination of antiviral drug,valacyclovir and its metabolite acyclovir in human plasma by a rapid liquid chromatography–tandem mass spectrometry method

A simple, sensitive and high throughput liquid chromatography/positive-ion electrospray ionization mass spectrometry (LC–ESI-MS/MS) method has been developed for the simultaneous determination of valacyclovir and acyclovir in human plasma using fluconazole as internal standard (IS). The method involved solid phase extraction of the analytes and IS from 0.5 mL human plasma with no reconstitution and drying steps (direct injection of eluate). The chromatographic separation was achieved on a Gemini C18 analytical column using isocratic mobile phase, consisting of 0.1% formic acid and methanol (30:70 v/v), at a flow-rate of 0.8 mL/min. The precursor → product ion transition for valacyclovir (m/z 325.2 → 152.2), acyclovir (m/z 226.2 → 152.2) and IS (m/z 307.1 → 220.3) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) mode. The method was validated over the concentration range 5.0–1075 ng/mL and 47.6–10225 ng/mL for valacyclovir and acyclovir respectively. The mean recovery of valacyclovir (92.2%), acyclovir (84.2%) and IS (103.7%) from spiked plasma samples was consistent and reproducible. The bench top stability of valacyclovir and acyclovir was extensively evaluated in buffered and unbuffered plasma. It was successfully applied to a bioequivalence study in 41 healthy human subjects after oral administration of 1000 mg valacyclovir tablet formulation under fasting condition.     

UHPLC method for the simultaneous determination of β-blockers, isoflavones and their metabolites in human urine

A rapid-resolution ultra high-performance liquid chromatography separation method (UHPLC) for the simultaneous determination of the following β-blockers: milrinone, sotalol, metoprolol, propranolol and carvedilol, and their metabolites: 5′-hydroxylphenyl-carvedilol, O-desmethylcarvedilol, 4-hydroxypropranolol, α-hydroxy-metoprolol, O-desmethyl-metoprolol; the following isoflavones: genistein, daidzein, glycitin, glycitein, puerarin and biochanin A; as well as their metabolites: dihydrogenistein, desmethylglycitein, 8-hydroxygenistein, daidzein-7,4′-diglucoside, 8-hydroxydaidzein, dihydrobiochanin A in human urine was optimized. The analysed compounds were extracted from human urine by means of solid phase extraction (SPE). The effective UHPLC separation of the examined compounds was applied on a Hypersil GOLD? (50 mm × 2.1 mm, 1.9 μm) column with a gradient mobile phase system and a UV detector. The complete separation of all analytes was achieved within 8.0 min. The method was validated for the determination of the aforementioned substances in human urine. The linear ranges, limits of detection (LOD) and limits of quantification (LOQ) for β-blockers, isoflavones and their metabolites were determined. The intra- and inter-day precision (%C.V.) was less than 4.48%, and the intra-day and inter-day accuracy was less than 4.74%. The tested SPE sorbent proved that appropriate absolute recoveries can be obtained for Oasis HLB (Waters). The mean recovery of the analytes, using the new SPE procedure, amounted from 70.14% to 99.85%. The present paper reports, for the first time, the method for the determination of β-blockers, isoflavones and their metabolites in human urine samples. The newly developed method was suitably validated and successfully applied for the analysis of the certain of the aforementioned analytes in human urine samples obtained from the patients suffering cardiovascular disease.     

High performance liquid chromatographic determination of plasma free and total tazobactam and piperacillin

A high-pressure liquid chromatography (HPLC) method with ultraviolet detection was developed for the measurement of plasma free and total tazobactam and piperacillin. This method is simple and fast, requiring only 11 min for the HPLC run and a sample preparation of about 11 min for total drugs and 10 min for free drugs. The procedure for the assay involves the treatment of plasma with acetonitrile for total drugs determination, and the use of a centrifugal filter device to deproteinize plasma for free drugs determination. The HPLC column, a Hypersil-ODS, was equilibrated with an eluent mixture composed of acetonitrile–potassium phosphate (pH 2.6). CVs for repeatability of tazobactam and piperacillin measurements ranged from 4.30 to 6.60; CVs for reproducibility ranged from 5.60 to 9.40. Mean analytical recoveries ranged from 100.4 to 103%. A linear relationship was obtained between peak area and drugs concentration in the range studied (0–62.5 mg/L for tazobactam and 0–500 mg/L for piperacillin). The equation for regression line were y = 19x ? 1.4 for tazobactam and y = 1.7x ? 0.9 for piperacillin; correlation coefficients were >0.999. The lower limit of quantitation (LLQ) for standard samples was about 0.12 mg/L for tazobactam and 0.49 mg/L for piperacillin, respectively. The lower limit of detection (LLD) was 0.06 mg/L for tazobactam and 0.24 mg/L for piperacillin. This HPLC assay for tazobactam and piperacillin is sensitive and accurate, and provides a reliable determination of both free and total tazobactam and piperacillin in human plasma, thus allowing the determination of these analytes in patients receiving tazocillin therapy.     

Optimized method for the determination of itopride in human plasma by high-performance liquid chromatography with fluorimetric detection

A high-performance liquid chromatographic method with fluorescence detection for the determination of itopride in human plasma is reported. The sample preparation was based on liquid–liquid extraction of itopride from plasma with t-butylmethylether and dichloromethane (70:30, v/v) mixture followed by a back extraction of the analyte to the phosphate buffer (pH 3.2). Liquid chromatography was performed on an octadecylsilica column (55 mm × 4 mm, 3 μm particles), the mobile phase consisted of acetonitrile–triethylamine–15 mM dihydrogenpotassium phosphate (14.5:0.5:85, v/v/v), pH of the mobile phase was adjusted to 4.8. The run time was 3 min. The fluorimetric detector was operated at 250/342 nm (excitation/emission wavelength). Naratriptan was used as the internal standard. The limit of quantitation was 9.5 ng/ml using 0.5 ml of plasma. The method precision and inaccuracy were less than 8%. The assay was applied to the analysis of samples from a bioequivalence study.     

Quantitation of polar analytes using column-switching: Application to oxycodone and three metabolites in human plasma

We present herein a sensitive and selective assay for the determination of oxycodone and its main metabolites, oxymorphone, noroxycodone and noroxymorphone in human plasma, using column-switching and liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). Sample preparation comprised protein precipitation with perchloric acid. After neutralization, the supernatant was injected without any evaporation step onto a polymeric, pH-resistant cartridge (HySphere Resin GP 10–12 μm) for sample clean-up (Prospekt II). The latter operation was achieved by using alkaline conditions to ensure retention of analytes and methanol for matrix interference removal. More than two hundred plasma samples could be analyzed with a single cartridge. Analytes were desorbed in the backflush mode and were separated on a conventional reversed phase column (XTerra MS 4.6 × 50 mm, 3.5 μm), using an acidic mobile phase (i.e. containing 0.1% of formic acid). Mass spectrometric detection was achieved with a 4000 Q TRAP equipped with an atmospheric pressure chemical ionization (APCI) source, in positive ionization mode, operated in the selected reaction monitoring mode (SRM). Starting from a plasma volume of 250 μl, quantification ranges were 25–10,000 pg/ml for OXM and NOXM and 50–10,000 pg/ml for OXC and NOXC. Accuracy was found to be within 98% and 108% and precision better than 7%. Replicate determination of incurred or study samples ensured the method to be reproducible and usable for clinical studies.     

A sensitive LC–MS/MS method for quantification of a nucleoside analog in plasma: Application to in vivo rat pharmacokinetic studies

A LC–MS/MS method was developed and validated for determination of nucleoside analog (NA) in rat plasma. The method run time was 6 min and the limit of quantification (LOQ) was estimated at 100 pg/mL. The extraction procedure consisted on plasma samples protein precipitation with an acetonitrile solution which contained the stable isotope labeled internal standard (IS). Chromatography was performed on a newly developed C₁₆ column (150 mm × 4.6 mm, 5 μm) in order to avoid the use ion pair salts. The samples were eluted at 0.8 mL/min with a gradient of mobile phase made of water and acetonitrile both acidified with 0.5% acetic acid and 0.025% trifluoroacetic acid (TFA). A tandem mass spectrometer was used as a detector for quantitative analysis. Intra-run and inter-run precision and accuracy within ±15% were achieved during a 3-run validation for quality control samples at four concentration levels in rat plasma, over a concentration ranging between 0.1 and 1000 ng/mL. The data indicate that our LC–MS/MS assay is an effective method for the pharmacokinetics study of NA in rat plasma.     

Optimization of a rapid microwave-assisted extraction method for the simultaneous determination of opiates,cocaine and their metabolites in human hair

A rapid and cleanup-free microwave-assisted extraction (MAE) method is proposed for the simultaneous extraction of six illegal drugs of abuse – cocaine, benzoylecgonine (BZE), cocaethylene (CCE), morphine, 6-monoacethylmorphine (6AM) and codeine – from human hair samples. The analytes were determined using high performance liquid chromatography (HPLC) with photodiode array UV detection. The influence of several variables on the efficiency of the MAE procedure was investigated in detail by a multi-objective optimization approach based on a hybrid experimental design (17 experiments) and desirability functions. Six drugs were successfully extracted from human hair with recoveries close to 100% and good reproducibility (<3.6% RSD) under the optimal MAE conditions: 11 mL dichloromethane (DCM) extraction solvent, 60 °C extraction temperature, 9 min extraction time and 0.5 mL of methanol (MeOH) added to 50 mg of the hair sample in the extraction vessels. Limits of quantification of 0.2 ng mg^?1 were found for the studied compounds. A comparison of sample preparation procedures, including MAE, enzymatic digestion and digestion by aqueous acids, was also conducted. The results indicated that the global behaviour of sample procedures provided similar satisfactory recoveries ranging from 86 to 100%. Indeed, the MAE procedure resulted in a reduction of extraction time by 100-fold and the elimination of cleanup steps. Slightly higher recoveries of morphine, 6AM, BZE and CCE, at 1 ng mg^?1 concentration level and cocaine at 40 ng mg^?1 concentration level, were achieved using MAE. Lastly, the proposed MAE method was applied to several human hair samples from multidrug abusers.     

Simultaneous determination of ABT-888, a poly (ADP-ribose) polymerase inhibitor,and its metabolite in human plasma by liquid chromatography/tandem mass spectrometry

A reversed-phase liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous determination of ABT-888 and its major metabolite (M8) in human plasma. Sample preparation involved a liquid–liquid extraction by the addition of 0.25 ml of plasma with 10 μl of 1 M NaOH and 1.0 ml ethyl acetate containing 50 ng/ml of the internal standard zileuton. The analytes were separated on a Waters XBridge C₁₈ column using a gradient mobile phase consisting of methanol/water containing 0.45% formic acid at the flow rate of 0.2 ml/min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the ABT-888 and M8 concentration ranges of 1–2000 ng/ml in human plasma. The lower limits of quantitation (LLOQ) were 1 ng/ml for both ABT-888 and M8 in human plasma. The accuracy and within- and between-day precisions were within the generally accepted criteria for bioanalytical method (<15%). This method was successfully employed to characterize the plasma concentration–time profile of ABT-888 after its oral administration in cancer patients.     

Online solid phase extraction with liquid chromatography–tandem mass spectrometry to analyze remoxipride in small plasma-, brain homogenate-, and brain microdialysate samples

Remoxipride is a selective dopamine D₂ receptor antagonist, and useful as a model compound in mechanism-based pharmacological investigations. To that end, studies in small animals with serial sampling over time are needed. For these small volume samples currently no suitable analytical methods are available. We propose analytical methods for the detection of low concentrations remoxipride in small sample volumes of plasma, brain homogenate, and brain microdialysate, using online solid phase extraction with liquid chromatography–tandem mass spectrometry. Method development, optimization and validation are described in terms of calibration curves, extraction yield, lower limit of quantification (LLOQ), precision, accuracy, inter-day- and intra-day variability. The 20 μl plasma samples showed an extraction yield of 76%, with a LLOQ of 0.5 ng/ml. For 0.6 ml brain homogenate samples the extraction yield was 45%, with a LLOQ of 1.8 ng/ml. The 20 μl brain microdialysate samples, without pre-treatment, had a LLOQ of 0.25 ng/ml. The precision and accuracy were well within the acceptable 15% range. Considering the small sample volumes, the high sensitivity and good reproducibility, the analytical methods are suitable for analyzing small sample volumes with low remoxipride concentrations.     

A novel validated procedure for the determination of nicotine,eight nicotine metabolites and two minor tobacco alkaloids in human plasma or urine by solid-phase extraction coupled with liquid chromatography–electrospray ionization–tandem mass spectrometry

A novel validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) procedure was developed and fully validated for the simultaneous determination of nicotine-N-β-d-glucuronide, cotinine-N-oxide, trans-3-hydroxycotinine, norcotinine, trans-nicotine-1′-oxide, cotinine, nornicotine, nicotine, anatabine, anabasine and cotinine-N-β-d-glucuronide in human plasma or urine. Target analytes and corresponding deuterated internal standards were extracted by solid-phase extraction and analyzed by LC–MS/MS with electrospray ionization (ESI) using multiple reaction monitoring (MRM) data acquisition. Calibration curves were linear over the selected concentration ranges for each analyte, with calculated coefficients of determination (R²) of greater than 0.99. The total extraction recovery (%) was concentration dependent and ranged between 52–88% in plasma and 51–118% in urine. The limits of quantification for all analytes in plasma and urine were 1.0 ng/mL and 2.5 ng/mL, respectively, with the exception of cotinine-N-β-d-glucuronide, which was 50 ng/mL. Intra-day and inter-day imprecision were ≤14% and ≤17%, respectively. Matrix effect (%) was sufficiently minimized to ≤19% for both matrices using the described sample preparation and extraction methods. The target analytes were stable in both matrices for at least 3 freeze–thaw cycles, 24 h at room temperature, 24 h in the refrigerator (4 °C) and 1 week in the freezer (?20 °C). Reconstituted plasma and urine extracts were stable for at least 72 h storage in the liquid chromatography autosampler at 4 °C. The plasma procedure has been successfully applied in the quantitative determination of selected analytes in samples collected from nicotine-abstinent human participants as part of a pharmacokinetic study investigating biomarkers of nicotine use in plasma following controlled low dose (7 mg) transdermal nicotine delivery. Nicotine, cotinine, trans-3-hydroxycotinine and trans-nicotine-1′-oxide were detected in the particular sample presented herein. The urine procedure has been used to facilitate the monitoring of unauthorized tobacco use by clinical study participants at the time of physical examination (before enrollment) and on the pharmacokinetic study day.     

Determination of ractopamine and clenbuterol in feeds by gas chromatography–mass spectrometry

A simple, sensitive and reproducible gas chromatographic–mass spectrometric method was developed for monitoring ractopamine (RAC) and clenbuterol (CLB) in feeds. Feed samples were extracted with 0.1 M perchloric acid, centrifuged, neutralized, followed by liquid–liquid extraction with ethyl acetate-isopropanol (9:1, v/v). The concentrated extracts were dissolved in 0.02 M NH₄Ac (pH 5.2), and applied to a solid phase extraction SCX cartridge for cleanup. The drugs were eluted with 3% (v/v) ammonia hydroxide in methanol, and the eluate was evaporated to dryness. The residue was derivatized with N,O-bis (trimethylsilyl) trifluoroacetamide at 80 °C for 1 h, and cooled, then analyzed by gas chromatography–mass spectrometry. The selected ions monitoring mode was performed at m/z 179, 250, 267 and 502 for RAC, and m/z 86, 243, 262 and 277 for CLB. Recoveries of RAC and CLB from concentrated feeds and premix fortified at 10, 100 and 5000 μg/kg were between 64.6 and 84.2%, with relative standard deviations of less than 15%. The limits of detection were about 4 μg/kg for RAC and 2 μg/kg for CLB.     

Speciation of organotin compounds in urine by GC–MIP-AED and GC–MS after ethylation and liquid–liquid extraction

A method for the determination of organotin compounds in urine samples based on liquid–liquid extraction (LLE) in hexane and gas chromatographic separation was developed and optimized. Seven organotin species, namely monobutyltin (MBT), dibutyltin (DBT), tributyltin (TBT), tetrabutyltin (TeBT), monophenyltin (MPhT), diphenyltin (DPhT) and triphenyltin (TPhT), were in situ derivatized by sodium tetraethylborate (NaBEt₄) to form ethylated less polar derivatives directly in the urine matrix. The critical parameters which have a significant effect on the yield of the successive liquid–liquid extraction procedure were examined, by using standard solutions of tetrabutyltin in hexane. The method was optimized for use in direct analysis of undiluted human urine samples and ways to overcome practical problems such as foam formation during extraction, due to various constituents of urine are discussed. After thorough optimization of the extraction procedure, all examined species could be determined after 3 min of simultaneous derivatization and extraction at room temperature and 5 min phase separation by centrifugation. Gas chromatography with a microwave-induced plasma atomic emission detector (MIP-AED) as element specific detector was employed for quantitative measurements, while a quadrupole mass spectrometric detector (MS) was used as molecular specific detector. The detection limits were between 0.42 and 0.67 μg L^?1 (as Sn) for the quantitative LLE–GC–MIP-AED method and the precision between 4.2% and 11.7%, respectively.     

Improved method for the determination of cyclic guanosine monophosphate (cGMP) in human plasma by LC–MS/MS

Cyclic guanosine monophosphate (cGMP) is an important second messenger molecule involved in gating ion channels and activating protein kinases. Here, we describe a validated LC–MS/MS method for the quantification of cGMP in human plasma, utilizing a stable isotope labeled analogue of cGMP as I.S. Plasma samples were extracted and concentrated by weak anion exchange solid phase extraction and the extracts were chromatographically separated on a porous graphitic carbon column. The analytes were detected by positive electrospray ionization and tandem mass spectrometry. The calibration function was linear in the range 1–20 nM and the intra- and inter-day precision showed relative standard deviations of better than 2 and 6%, respectively. The accuracy was always better than 4%. Plasma concentrations in healthy human subjects determined with this method were 3.92 ± 1.17 nM (n = 20). The method was, due to its isotope labeled I.S., matrix independent.     

Elimination of diastereomer interference to determine Telcagepant (MK-0974) in human plasma using on-line turbulent-flow technology and off-line solid-phase extraction coupled with liquid chromatography/tandem mass spectrometry

To eliminate the diastereomer interference on Telcagepant (MK-0974) determination during clinical study support, on-line high turbulent-flow liquid chromatography (HTLC) methods, HTLC-A and HTLC-B that covered dynamic range of 0.5–500 nM and 5–5000 nM, respectively, were developed. To meet the requirement of rapid assay transfer among multiple laboratories and analysts, a solid-phase extraction (SPE) assay was derived from the existing HTLC-B assay under the same dynamic range. The on-line HTLC assays were achieved through direct injection of plasma samples, extraction of analyte with a Cohesive C₁₈ column (50 mm × 0.5 mm, 50 μm), followed by HPLC separation on a FluoPhase RP column (100 mm × 2.1 mm, 5 μm) and MS/MS detection. The off-line SPE assay used Waters Oasis^®HLB μElution plate to extract the analytes from plasma matrix before injecting on a FluoPhase RP column (150 mm × 2.1 mm, 5 μm) for LC–MS/MS analysis. Under both on-line and off-line assay conditions, the diastereomer 1c was chromatographically separated from MK-0974. Cross-validation with the pooled samples demonstrated that both on-line and off-line assays provided comparable data with a difference of <2.6%. The assays were proved to be specific, accurate and reliable, and have been used to support multiple clinical studies. The pros and cons of on-line and off-line assays with regard to man power involved in sample preparation, total analysis time, carryover, cost efficiency, and the requirement for assay transfer are discussed.     

Plasma and cerebral spinal fluid tranexamic acid quantitation in cardiopulmonary bypass patients

A method for the determination of tranexamic acid (TXA) in human plasma and cerebral spinal fluid (CSF) was developed. Analyses were performed by ultra performance liquid chromatography with tandem mass spectrometry detection (UPLC–MS/MS) using ?-aminocaproic acid (ACA) as an internal standard. TXA and ACA were extracted from a 50 μL sample of plasma or CSF using a methanol protein crash protocol, and chromatographic separation was performed on an ACQUITY? TQD mass spectrometer using a UPLC C18 BEH 1.7 μm column with a water and methanol gradient containing 0.1% formic acid. The detection and quantitation was performed by positive ion electrospray ionization using the multiple reaction monitoring (MRM) mode. The method was linear over the concentration range of 0.1–10.0 μg/mL, with lower limit of quantitation of 0.1 μg/mL for TXA. The intra- and inter-assay precision was less than 12% and 13% respectively at the plasma and CSF TXA concentrations tested. The present method provides a relatively simple and sensitive assay with short turn-around-time. The method has been successfully applied to assess the plasma and CSF concentrations of tranexamic acid achieved with only one dosing regimen of tranexamic acid in patients undergoing cardiopulmonary bypass surgery (CPB).     

Validation of an optimized method for the determination of iodine in human breast milk by inductively coupled plasma mass spectrometry (ICPMS) after tetramethylammonium hydroxide extraction

In this study a novel method to determine iodine concentrations in human breast milk was developed and validated. The iodine was analyzed by inductively coupled plasma mass spectrometry (ICPMS) following tetramethylammonium hydroxide (TMAH) extraction at 90 °C in disposable polypropylene tubes. While similar approaches have been used previously, this method adopted a shorter extraction time (1 h vs. 3 h) and used antimony (Sb) as the internal standard, which exhibited greater stability in breast milk and milk powder matrices compared to tellurium (Te). Method validation included: defining iodine linearity up to 200 μg L⁻¹; confirming recovery of iodine from NIST 1549 milk powder. A recovery of 94–98% was also achieved for the NIST 1549 milk powder and human breast milk samples spiked with sodium iodide and thyroxine (T4) solutions. The method quantitation limit (MQL) for human breast milk was 1.6 μg L⁻¹. The intra-assay and inter-assay coefficient of variation for the breast milk samples and NIST powder were <1% and <3.5%, respectively. NIST 1549 milk powder, human breast milk samples and calibration standards spiked with the internal standard were all stable for at least 2.5 months after extraction. The results of the validation process confirmed that this newly developed method provides greater accuracy and precision in the assessment of iodine concentrations in human breast milk than previous methods and therefore offers a more reliable approach for assessing iodine concentrations in human breast milk.     

Identification and quantitative determination of a major circulating metabolite of gambogic acid in human

Gambogic acid (GA), a promising anticancer candidate, is a polyprenylated xanthone abundant in the resin of Garcinia morella and Garcinia hanburyi. The major circulating metabolite of GA in human, 10-hydroxygambogic acid (10-OHGA), was identified by comparison of the retention time and mass spectra with those of reference standard using liquid chromatography–tandem mass spectrometry. The reference standard of 10-OHGA was isolated from bile samples of rats after intravenous injection of GA injection, and its structure was confirmed by NMR. Then, a selective and sensitive method was developed for the quantitative determination of this metabolite in human plasma. After liquid–liquid extraction by ethyl acetate, the analyte and the internal standard were separated on a Sepax HPC18 column (100 mm × 2.1 mm i.d., 3.0 μm) with a mobile phase of 10 mM ammonium acetate water solution containing 0.1% formic acid–acetonitrile (20:80, v/v). The detection was performed on a single quadrupole mass spectrometer equipped with electrospray ionization (ESI) source. The calibration curve was linear over the range of 3–2000 ng/mL for 10-OHGA. The developed quantification method can now be used for the pharmacokinetic and pharmacological studies of 10-OHGA after intravenous infusion of GA injection in human.