Analysis of methanol or formic acid in body fluids by headspace solid-phase microextraction and capillary gas chromatography

Methanol and its metabolite formic acid have been found extractable from human whole blood and urine by headspace solid-phase microextraction (SPME) with a Carboxen/polydimethylsiloxane fiber. The headspace SPME for formic acid was carried out after derivatization to methyl formate under acidic conditions. The determinations of both compounds were made by using acetonitrile as internal standard (IS) and capillary gas chromatography (GC) with flame ionization detection. The headspace SPME–GC gave sharp peaks for methanol, methyl formate and I.S.; and low background noises for whole blood and urine samples. Extraction efficiencies were 0.25–1.05% of methanol and 0.38–0.84% formic acid for whole blood and urine. The calibration curves for methanol and formic acid showed excellent linearity in the range of 1.56 to 800 and 1.56 to 500 μg/0.5 ml of whole blood or urine, respectively. The detection limits were 0.1–0.5 μg/0.5 ml for methanol and 0.6 μg/0.5 ml for formic acid for both body fluids. The within-day relative standard deviations in terms of extraction efficiency for both compounds in whole blood and urine samples were not greater than 9.8%. By using the established SPME method, methanol and formic acid were successfully separated and determined in rat blood after oral administration of methanol.     

Identification of N‐octylnortadalafil and its Stereoisomers in Dietary Supplements with Chiral Liquid Chromatography–Circular Dichroism

A direct chiral liquid chromatography–circular dichroism (LC‐CD) method was developed for the simple and rapid identification of N‐octylnortadalafil [(6R, 12aR)‐6‐(1,3‐benzodioxol‐5‐yl)‐2‐octyl‐2,3,6,7,12,12a‐hexahydropyrazino[1’,2’:1,6]pyrido[3,4‐b]indole‐1,4‐dione; RR‐OTDF] and its stereoisomers in dietary supplements. Samples were extracted with methanol. Compounds were then separated by chiral LC‐CD using Chiralcel OD‐RH (4.6 × 1 50 mm, 5 µm) with 5 mM ammonium formate (pH 3)/0.1% formic acid in acetonitrile (95:5, v/v) mixture solution (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The isocratic elution used was mobile phase A / mobile phase B (3:7, v/v) at a flow rate of 1 ml/min. The column temperature was held at 30°C. RR‐OTDF and its stereoisomers were separated within 20 min with the resolution factors being over 2.0. Using this method, RR‐OTDF and (6R, 12aS)‐6‐(1,3‐benzodioxol‐5‐yl)‐2‐octyl‐2,3,6,7,12,12a‐hexahydropyrazino[1’,2’:1,6]pyrido[3,4‐b]indole‐1,4‐dione were detected in a dietary supplement. Chirality 28:204–208, 2016. © 2016 Wiley Periodicals, Inc.     

Mechanism of formaldehyde biodegradation by Pseudomonas putida

Summary Formaldehyde biodegradation by a strain of Pseudomonas putida has been studied. The results indicate that this biodegradation is initiated by a dismutation reaction, yielding as products formic acid and methanol. The degradation of methanol and formic acid begins after exhaustion of formaldehyde in the medium, and presents a diauxic pattern: first formic acid is consumed followed by methanol. Moreover, cell viability, which is affected by the amount of added formaldehyde, has been determined. Offprint requests to: N. Adroer     

Convenient preparation of L-arabino-hexos-5-ulose derivatives from lactose

2,6-di-O-benzyl- (9), 2-O-benzyl-3,4-O-isopropylidene- (19), and 2-O-benzyl-6-O-m-chlorobenzoyl-L-arabino-hexos-5-ulose (20) have been prepared using 4'-deoxy-4'-eno- and 6'-deoxy-5'-eno lactose dimethyl acetal derivatives 7 and 14 as key intermediates. The synthesis of enol ethers 7 and 14 has been performed with good yields by base-promoted elimination of acetone or p-toluenesulfonic acid from 2',6'-di-O-benzyl-, and 6'-O-p-toluenesulfonyl-2,3:5,6:3',4'-tri-O-isopropylidenelactose dimethyl acetal, respectively. The epoxidation with MCPBA of 7 and 14 in methanol or dichloromethane furnishes C-5'-methoxy and C-5'-m-chlorobenzoyloxy derivatives, easily transformed with good yields into L-arabino 5-ketoaldohexoses 9, 19 and 20.     

Synthesis of the amino acid conjugates of <Emphasis Type="Italic">epi</Emphasis>-jasmonic acid

The TES ether of the C6-hydroxy derivative of naturally occurring epi-jasmonic acid (epi-JA) was designed as epimerization-free equivalent of epi-JA. The TES ether was synthesized from (1R,4S)-4-hydroxycyclopent-2-enyl acetate in 13 steps. The acid part of the ether was activated with ClCO₂Bu ⁱ and subjected to condensation with l-amino acid at room temperature for 48 h. The TES group in the condensation product was removed in HCO₂H (0°C, 30 min) and the resulting hydroxyl group was oxidized with Jones reagent (acetone, 0°C, 30 min) to furnish the amino acid conjugate of epi-JA. The amino acids examined are l-isoleucine, l-leucine, l-alanine, l-valine, and d-allo-isoleucine, which afforded the conjugates in 48–68% yields with 89–96% diastereomeric purity over the trans isomers. Similarly, the possible three stereoisomers of epi-JA were condensed with l-isoleucine successfully, producing the corresponding stereoisomers in good yields.     

Rapid method for simultaneous determination of 20 components in Isodon nervosa by high‐performance liquid chromatography–electrospray ionisation tandem mass spectrometry

Introduction – Isodon nervosa is a commonly used traditional Chinese medicine including diterpenoids, phenolic acids, triterpenoids and volatile oil. Qualitative and quantitative analysis of multi‐components is important for its quality control. Objective – To establish a liquid chromatography–electrospray ionisation–mass spectrometry method for simultaneous analysis of 20 bioactive constituents of Isodon nervosa in different places of China and different parts of this herb. Methodology – The optimal chromatographic conditions were achieved on a C₁₈ column (250 × 4.6 mm, 5 µm) with with linear gradient elution with 0.1% aqueous formic acid : methanol containing 0.1% formic acid at a flow‐rate of 0.7 mL/min in 15 min. The identification and quantification of those analytes were achieved on a hybrid quadrupole linear ion trap mass spectrometer. Multiple‐reaction monitoring scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. Full validation of the method was carried out (linearity, precision, accuracy, limit of detection and limit of quantification). Results – The results indicated that the method was simple, rapid, specific and reliable. The proposed method was successfully applied for the qualitative and quantitative analysis of 20 chemical compositions in Isodon nervosa samples. Conclusion – Twenty chemical compositions in 21 batches of wild and cultivated Isodon nervosa samples from different sources had great variation in the contents. Copyright © 2010 John Wiley & Sons, Ltd.     

Simple Method for Analyzing Phosphoamino Acids by Thin-layer Chromatography: Application to Elongation Factor 1 from Wheat Embryo

A simple and rapid method was developed separating phosphoserine, phosphothreonine, phosphotyrosine, phosphoric acid, and ATP by one-dimensional cellulose thin-layer chromatography, using a freshly prepared solvent system of 1-butanol–formic acid–water (5:2:1). This system was applied to determine phosphoamino acid (phosphoserine) of wheat elongation factor 1β phosphorylated by casein kinase II from the wheat embryo.     

Solution properties and structure of brain proteolipids

Bovine white matter proteolipids have been studied by several physical methods and have been found to exist as micelles in 2 : 1 (v/v) chloroform–methanol solution. The data would indicate the existence of a critical micelle concentration at 0.017–0.022 g/100 ml. The curve appears linear in the range 0.017–0.2 g/100 ml, but from the data at higher concentrations it would appear that a change in slope is occurring in the region 0.2–0.3 g/100 ml. Light-scattering measurements on 2 : 1 (v/v) chloroform–methanol solutions containing more than 0.2 g/100 ml of proteolipid yielded a weight-average aggregate weight of 2.9 × 10⁶and a radius of gyration of 64.5 Å. The intrinsic viscosity of the solutions was 0.32 dl/g and the Huggins constant was 1.085. Light-scattering measurements in 88.5% formic acid–0.5M sodium formate yielded a weight-average aggregate weight of 7.1 × 10⁶ and a radius of gyration of 241 Å. The intrinsic viscosity observed for this solvent system is 0.14 dl/g and the Huggins constant is 1.005. Osmotic pressure measurements in 2 : 1 (v/v) chloroform–methanol containing less than 0.2 g/ 100 ml of proteolipid yielded a number-average aggregate weight of 7.2 × 10⁴ Ultracentrifugal analysis in 1.5:1 (v/v) methylene chloride–methanol showed two broad peaks with, s values of s_1.5% = 20.05 S, s_2% = 19.79 S for the minor peak and s_1.5%,2% = 1.86 S for the major peak. Optical rotatory dispersion studies revealed large changes in b₀ with change in solvent and proteolipid concentration. The present data suggest that the mode of attachment of protein to lipid is primarily of a noncovalent type. The results of this investigation also suggest that the proteolipid micelle above 0.2 g/100 ml is cylindrical (prolate ellipsoid) in 2:1 (v/v) chloroform-methanol and approaches a more spherical shape in 88.5% formic acid. A structure for the proteolipid micellar complex above concentrations of 0.2 g/100 ml is proposed.     

Formylation of primary hydroxyl groups in sugars

Application of 99% formic acid at 25 degrees formylates primary hydroxyl groups as shown by the easy formation of 6-O-formyl-D-glucopyranose and 6-O-formyl-D-fructofuranose, isolated as their tetra-acetates in yields of 77% and 31%, respectively. Likewise, D-glucitol gave the 2,3,4,5-tetra-O-acetyl-1,6-di-O-formyl derivative (84%) and methyl alpha-D-glucopyranoside gave the 6-formate (74%) characterized as the crystalline triacetate. On storage of the triacetate in methanol at 25 degrees, the formyl group was lost and the 2,3,6-triacetate was formed, whereas the corresponding tribenzoate was deformylated in acidic methanol without migration of the benzoyl groups.     

Methanol conversion in Eubacterium limosum

The conversion of methanol by cell-free extracts of the acetogenic bacterium Eubacterium limosum was studied. Incubation of mixed cell-free extracts of both E. limosum and Methanobacterium formicicum resulted in methane formation from methanol in the presence of ATP and 2-mercaptoethanesulfonic acid. The separate extracts were not able to perform this reaction. Addition of ferredoxin obtained from Methanosarcina barkeri to the mixed extracts resulted in increased methane formation. The enzyme, responsible for methanol binding in cell-free extract of E. limosum, was inactivated by FAD under N₂ and exhibited maximal activity under an atmosphere of H₂. This enzyme contains a firmly bound cobalamin which was methylated by methanol in the presence of ATP. It was demethylated in the presence of methylcobalamin: coenzyme M methyltransferase obtained from M. barkeri under concomitant formation of methylated coenzyme M. These properties are similar to those of methanol: 5-hydroxybenzimidazolylcobamide methyltransferase from M. barkeri. It was proposed that methylotrophic acetogens and methylotrophic methanogens use similar enzymes in the first step of methanol conversion.Abbreviations HS-CoM 2-mercaptoethanesulfonic acid - CH₃S-CoM 2-(methylthio)ethanesulfonic acid - BrES 2-bromoethanesulfonic acid - TES N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid - MT₁ methanol: 5-hydroxybenzimidazolylcobamide methyltransferase - MT₂ methylcobalamin - HS-CoM methyltransferase - DMBI 5,6-dimethylbenzimidazole and HBI, 5-hydroxybenzimidazole, are -ligands of corrinoids - (S-CoM)₂ 2,2-dithiodiethanesulfonic acid     

Simultaneous quantification of 14 bioactive constituents in Forsythia Suspensa by liquid chromatography–electrospray ionisation–mass spectrometry

Introduction – Forsythia suspensa is a commonly used traditional Chinese medicine including phenylethanoid glycosides, lignans, flavonoids, terpenes and volatile oils. Quantification of multi‐components is important for the quality control of Forsythia suspensa. Objective – To establish a liquid chromatography–electrospray ionisation–mass spectrometry method for simultaneous quantification of 14 bioactive constituents of Forsythia suspensa in different places of China and different parts of this herb. Methodology – The optimal chromatographic conditions were achieved on a Kromasil C₁₈ column (150 ¥ 4.6 mm, 5 mm) with gradient elution of methanol, acetonitrile and 0.1% formic acid in 27 min. Detection was performed in negative ionisation mode by monitoring the precursor–product combination in multiple reaction monitoring (MRM) mode. The validation of the method included tests of linearity, sensitivity, precision, repeatability, stability and accuracy. Results – All calibration curves showed good linear regression (r > 0.9990) within test ranges. The established method showed good precision and accuracy with overall intra‐day and inter‐day variations of 0.7–4.3 and 1.1–3.9% respectively, and overall recoveries of 96.65–101.2% for the compounds analysed. Conclusion – The proposed method was successfully applied for the quantitative analysis of 14 constituents in 12 Forsythia suspensa samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Aqueous and ethanolic extracts of Galinsoga parviflora and Galinsoga ciliata. Investigations of caffeic acid derivatives and flavonoids by HPTLC and HPLC-DAD-MS methods

Galinsoga ciliata Raf. Blake like Galinsoga parviflora Cav., comes from the Andes region. The chemical composition, activity and use are similar for both species. Galinsoga species are used in folk medicine as anti-inflammatory agents and accelerators for wound healing. Extracts are applied topically onto the skin to treat dermatological diseases, eczemas, lichens and hard-healing-wounds, and also to treat snakebites. Orally they used to cure flu and colds.In the studies using HPTLC method, different stationary phases, including unmodified silica gel, silica gels modified with CN, NH₂, DIOL and RP18 groups were tried. The best separation of the tested compounds was achieved on silica gel plates, when as mobile phases mixtures – ethyl acetate–acetic acid–formic acid–water (100:11:11:26, v/v/v/v), ethyl acetate–methanol–formic acid–water (50:3:4:6, v/v/v/v) and ethyl acetate–methyl ethyl ketone–formic acid–water (30:9:3:3, v/v/v/v) – were used. Using reference substances, in the examined extracts the presence of flavonoids: patulitrin, quercimeritrin, quercitagetrin, and phenolic acids – caffeic and chlorogenic acids was found.HPLC analyses of extracts were carried out on a reversed-phase Zorbax SB column (150 mm × 2.1 mm, 1.9 μm). The mobile phase (A) was water/acetonitrile/formic acid (95:5:0.1, v/v/v) and the mobile phase (B) was acetonitrile/formic acid (100:0.1, v/v). A linear gradient system was used: 0–30 min, 1–30% B. Application of HPLC-DAD-MS method confirmed the results obtained by HPTLC method. Moreover, in the tested extracts the presence of caffeoylglucaric acids as dominating compounds was detected.     

Studies on Mannan in Wood Pulp

The glucomannan isolated from holocellulose pulp of Akamatsu (Pinus densiflora Sieb. et Zucc.) as its triacetate was methylated and the following methylated sugars were obtained by hydrolysis: the 2,3,4,6-tetra-O-methyl ethers of d-glucose and d-mannose (I part) and the 2,3,6-tri-O-methyl ethers of d-mannose and d-glucose (34–37 parts). Periodate oxidation of the glueomannan showed that 1.00 mole of periodate was consumed per mole of hexose unit and 3 moles of formic acid liberated for every 33 hexose units.     

Methanol formation during lignin degradation by Phanerochaete chrysosporium

Summary Methanol formation during the degradation of synthetic lignin (DHP), spruce and birch milled wood lignin (MWL) by Phanerochaete chrysosporium Burds. was studied under different culture conditions. When 100-ml flasks with 15–20 ml volumes of culture media containing high glucose and low nitrogen concentrations were used the metabolism of methanol to formaldehyde, formic acid and CO₂ was repressed thereby facilitating methanol determination. In standing cultures with oxygen flushing the fungus converted up to 25% of the DHP-methoxyl groups to methanol and 0.5–1.5% to ¹⁴CO₂ within 22–24 h. Methanol formation from methoxyl-labelled DHP was strongly repressed by high nitrogen in the medium, by addition of glutamic acid and by culture agitation. These results indicate that methanol is formed only under ligninolytic conditions and during secondary metabolism. Methanol is most likely released both from the lignin polymer itself and from lignin degradation products. Methanol was also formed from MWL preparations with higher percentage yields produced from birch as compared to spruce MWL.Small amounts of methanol detected in cultures without lignin probably emanated from demethoxylation of veratryl alcohol synthesized de novo from glucose by the fungus during secondary metabolism. Catalase or superoxide dismutase added to the fungal culture prior to addition of lignin, did not decrease methanol formation. Horseradish peroxidase plus H₂O₂ in vitro caused 5–7% demethoxylation of O¹⁴CH₃-DHP in 22 h, while laccase gave smaller amounts of methanol (1.8%). Since addition of H₂O₂ gave similar results as peroxidase plus H₂O₂, it seems likely that the main effect of peroxidase demethoxylation emanates from the hydrogen peroxide.     

Effects of acetic and formic acid on ABE production by <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> and <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

The effect of acetic acid and formic acid on acetone-butanol-ethanol (ABE) production by solventogenic Clostridia was investigated. The ABE concentration in Clostridium acetobutylicum was found to have increased slightly on addition of 3.7 ∼ 9.7 g/L acetic acid, but was found to have drastically reduced in the presence of 11.7 g/L acetic acid. However, the solvent production of C. beijerinckii was not affected by addition of acetic acid in the range of 3.7 ∼ 11.7 g/L. C. acetobutylicum was more vulnerable to formic acid than C. beijerinckii. In C. acetobutylicum, the total ABE production decreased to 77% on addition of 0.4 g/L formic acid and 25% with 1.0 g/L formic acid. The total ABE production by C. acetobutylicum was also noted to have decreased from 15.1 to 8.6 g/L when 8.7 g/L acetic acid and 0.4 g/L formic acid co-existed. The solvent production by C. beijerinckii was not affected at all under the tested concentration range of formic acid (0.0 ∼ 1.0 g/L) and co-presence of acetic acid and formic acid. Therefore, C. beijerinckii is more favorable than C. acetobutylicum when the ABE is produced using lignocellulosic hydrolysate containing acetic and formic acid.     

Characterization of the metabolite produced by Mycosphaerella pinodes, the causal agent of mycosphaerella blight on field peas (Pisum sativum L.)

The metabolite produced by Mycosphaerella pinodes, the causal agent of mycosphaerella blight on field peas, was detected by thin layer chromatography (TLC) and was analyzed for its chemical and pathogenic characteristics. One blue dot was detected using 254nm UV light on TLC plate, and a spray of rho-anisaldehyde (110 degrees C, 30 min) also produced a blue dot. The solvent systems used for TLC analysis were ethyl acetate/water/acetone (5/2/5), chloroform/methanol/glacial acetic acid (19/10/2), toluene/ethyl acetate/90% formic acid (6/3/1), diethylether/methanol/water/90% formic acid (95/4/1/1), and bezene/methanol/acetic acid (24/2/1), with R(f) values (min-max) of 0.09-0.18, 0.88-0.95, 0.06-0.15, 0.39-0.47 and 0.05-0.12, respectively. The recovered metabolite from the TLC plate displayed UV absorption peaks at 212, 244, 250, 256 and 261 nm. The proposed formula of the main component of the metabolite was C16H12N3O6. The TLC-purified metabolite induced symptom of discoloration on detached pea leaves.     

Trifluoroethanol‐containing RP‐HPLC mobile phases for the separation of transmembrane peptides human glycophorin‐A,integrin alpha‐1, and p24: analysis and prevention of potential side reactions due to formic acid

Reversed‐phase high‐pressure liquid chromatography analysis and purification of three hydrophobic, aggregation‐prone peptides, composed mainly of the transmembrane (TM) sequence, were performed using elution systems containing 2,2,2‐trifluoroethanol (TFE). The addition of 10–16% TFE to a common mobile phase, such as a water/acetonitrile/propanol (PrOH) or a water/PrOH/formic acid system, markedly improved the chromatographic separation of these peptides. The superior performance of TFE‐containing systems in separating peptides over water/PrOH/formic acid systems [Bollhagen R. et al., J. Chromatogr. A, 1995; 711 : 181–186.] clearly demonstrated that adding TFE to the mobile phase is one of best methods for TM‐peptide purification. Characterization of the potential side reactions using MALDI and ESI‐LIT/Orbitrap mass spectrometry indicated that prolonged incubation of peptides in a mixture of TFE–formic acid possibly induces O‐formylation of the Ser residue and N‐formylation of the N‐terminus of peptides. The conditions for selective removal of the formyl groups from TM peptides were also screened. We believe that these results will expand our ability to analyze and prepare hydrophobic, aggregation‐prone TM peptides and proteins. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Simultaneous determination of ribavirin and ribavirin base in monkey plasma by high performance liquid chromatography with tandem mass spectrometry

For the first time, a liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the simultaneous determination of ribavirin and rabavirin base was developed and validated over the concentration range of 10-5,000 ng/ml, respectively, using a 0.025 ml monkey plasma sample. Ribavirin, ribavirin base, and the internal standards were extracted from monkey plasma via protein precipitation. After evaporation of the supernatant, the extract was reconstituted with 5% methanol (containing 0.1% formic acid) and injected onto the LC-MS/MS system. Optimum chromatographic separation was achieved on a Waters Atlantis dc18 (150 mm x 2.1mm, 5 microm) column with mobile phase run in gradient with 100% water containing 0.5% formic acid (A) and 90% acetonitrile (containing 0.5% formic acid (B). The flow rate was 0.4-0.6 ml/min with total cycle time of approximately 7.0 min. Post-column addition of acetonitrile (containing 0.1% formic acid) at 0.3 ml/min was used to increase the ionization efficiency in the MS source. The method was validated for sensitivity, linearity, reproducibility, stability and recovery. Lack of adverse matrix effect and carry-over was also demonstrated. The intra-day and inter-day precision and accuracy of the quality control (QC) samples were <9.0% relative standard deviation (R.S.D.) and 10.8% bias for ribavirin, and 10.3% R.S.D. and 11.3% bias for ribavirin base. The current specific, accurate and precise assay is useful in support of the toxicokinetic and pharmacokinetic studies of these compounds.     

Detoxification of hydrolysate by reactive-extraction for generating biofuels

We introduce a reactive extraction to detoxify hydrolysate before fermentation to biofuels. In the selection of diluents, n-octanol showed the highest removal yield of 5-hydroxymethylfurfural (5-HMF) and levulinic acid. The removal yields of inhibitors were normalized to 30-min reactions. In treatments with pure extractant or diluents, only 2 ～ 4.1% of the formic acid was removed. Tri-n-octylamine (extractant) removed levulinic acid and acetic acid more efficiently, and furfural was removed more efficiently than formic acid or 5-HMF. n-Octanol (polar diluent) removed levulinic acid and acetic acid, furfural, and 5-HMF at 21.2, 33.7, and 65.7%, respectively. In contrast, kerosene (inert diluent) only removed the furfural by 27.6%. Based on these results, the optimum reactiveextraction system comprised tri-n-octylamine as the extractant, n-octanol as the polar diluent, and kerosene as the inert diluent. The optimal proportion of complex extractant was 20% trialkylamine, 70% n-octanol, and 10% kerosene. By detoxification, 63.9% of acetic acid and levulinic acid, 24.4% of 5-HMF, 63.9% of formic acid, and 64.0% of furfural could be removed.     

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).