Determination of hydroxysafflor yellow A in biological fluids of patients with traumatic brain injury by UPLC‐ESI‐MS/MS after injection of Xuebijing

A simple, novel, specific, rapid and reproducible ultra‐performance liquid chromatography electrospray ionization tandem mass spectrometry method has been developed and validated for the determination of hydroxysafflor yellow A (HSYA) in biological fluids (plasma, urine and cerebrospinal fluid) of patients with traumatic brain injury after intravenous injection of Xuebijing (XBJ). Liquid–liquid extraction was performed, and separation was carried out on an Acquity UPLC? BEH C₁₈ column, with gradient elution using a mobile phase composed of methanol and 0.1% formic acid at a flow rate of 0.3 mL/min. A triple quadrupole tandem mass spectrometer with electrospray ionization was used for the detection of HSYA. The mass transition followed was m/z 611.0 → 491. The retention time was less than 3.0 min. The calibration curve was linear in the concentration range from 2 to 6125 ng/mL for cerebrospinal fluid, plasma and urine. The intra‐ and inter‐day precisions were <10%, and the relative standard deviation of recovery was <15% for HSYA in biological matrices. The method was successfully applied for the first time to quantify HSYA in the biological fluids (especially in cerebrospinal fluid) of patients with traumatic brain injury following intravenous administration of XBJ. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of ceftriaxone in cerebrospinal fluid by ion-pair liquid chromatography

An ion-pair liquid chromatographic assay was developed and validated for the determination of ceftriaxone in cerebrospinal fluid. Chromatographic separation was achieved on a C18 column (125 x 4 mm, 5 microm) with detection at 270 nm, a 1 mL/min flow rate and a 50 microL loop. The mobile phase consisted of 300 mL acetonitrile, 50 mL 0.1M phosphate buffer (pH 7.4), 3.2 g tetrabutylammonium bromide as the ion-pairing agent, and dilution with distilled deionized water to 1 L. Cephradine was used as the internal standard. The assay was linear for ceftriaxone concentrations of 0.5-50 microg/mL. The coefficients of variation for precision were <4.61%. The accuracy ranged from 96.07 to 102.42%. The detection and quantitation limits were 0.019 and 0.065 microg/mL, respectively. This method was used to quantify ceftriaxone in the cerebrospinal fluid of children with meningitis. The results showed that the method described here is useful for the determination of ceftriaxone in cerebrospinal fluid.     

Determination of paracetamol (acetaminophen) in different body fluids and organ samples after solid-phase extraction using HPLC and an immunological method

A solid-phase extraction method routinely used for serum samples was improved and applied to the qualitative and quantitative determination of paracetamol in different body fluids, e.g. blood, urine, cerebrospinal fluid, synovial fluid, vitreous humor, and in tissue samples. A very simple method showed best results: Body fluids were mixed with phenacetine as internal standard and phosphate buffer (pH 6.8). Then protein was precipitated using acetonitrile. After strong centrifugation the supernatant was transferred to a preconditioned Bakerbond C18-SPE-column. Elution with methanol without a prior washing step showed best recovery rates. The extracts were investigated using high-performance liquid chromatography with ultraviolet detection, a photometrical and an immunochemical method.     

Determination of paracetamol (acetaminophen) in different body fluids and organ samples after solid-phase extraction using HPLC and an immunological method

A solid-phase extraction method routinely used for serum samples was improved and applied to the qualitative and quantitative determination of paracetamol in different body fluids, e.g. blood, urine, cerebrospinal fluid, synovial fluid, vitreous humor, and in tissue samples. A very simple method showed best results: Body fluids were mixed with phenacetine as internal standard and phosphate buffer (pH 6.8). Then protein was precipitated using acetonitrile. After strong centrifugation the supematant was transferred to a preconditioned Bakerbond C18-SPE-column. Elution with methanol without a prior washing step showed best recovery rates. The extracts were investigated using high-performance liquid chromatography with ultraviolet detection, a photometrical and an immunochemical method.     

Mass fragmentographic determination of 4-hydroxy-3-methoxyphenylglycol (HMPG) in urine, cerebrospinal fluid, plasma and tissues using a deuterium-labelled internal standard.

4-Hydroxy-3-methoxyphenylglycol (HMPG) with the three hydrogen atoms in the side-chain replaced with deuterium (HMPG-D3) was used as the internal standard in the mass fragmentographic determination of free and conjugated HMPG in human urine, cerebrospinal fluid and plasma and in rat urine, liver and brain. HMPG-D3 was added to body fluids or homogenates followed by enzymatic hydrolysis of conjugates. HMPG was extracted with ethyl acetate, converted into the trifluoroacetyl derivative and analyzed by mass fragmentography. HMPG levels were 7.4 nmoles/ml plus or minus 2.8% in human urine, 73 pmoles/ml plus or minus 8.2% in human cerebrospinal fluid, 56 pmoles/ml plus or minus 5.4% in human plasma, 24 nmoles/ml plus or minus 3.6% in rat urine, 0.26 nmoles/g plus or minus 6.2% in rat brain and 99 pmoles/g plus or minus 13% in rat liver. The method is highly specific and sensitive, permitting analysis in small samples or in plasma and in tissues for which previously no methods for HMPG analysis were available.     

Analysis of circulating immune complexes isolated from plasma, cerebrospinal fluid and urine.

The protein nature of soluble immune complexes (IC) from fresh plasma, cerebrospinal fluid (CSF), and urine was studied by combining several analytical and biochemical techniques. In plasma and CSF, free immunoglobulins G were separated from larger IC by gel filtration with a fast protein liquid chromatographic system. In urine, IC were separated by precipitation with polyethylene glycol. IC were further purified by protein-A and protein-G affinity chromatography and analyzed by two-dimensional gel electrophoresis. Apart from plasma samples from healthy donors, IC from cases with macrocreatine kinase type 1 and multiple sclerosis were analyzed. For CSF two cases of multiple sclerosis and for urine one case with urinary tract infection are shown. The method can be used for the examination of IC of unknown protein composition in body fluids.     

Determination of cefotaxime and desacetylcefotaxime in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the analysis of the anti-bacterial agent cefotaxime and desacetylcefotaxime in physiological fluids. Plasma or serum samples were mixed with chloroform--acetone to remove proteins and most lipid material. The aqueous phase was then freeze-dried, reconstituted in mobile phase and chromatographed on a reversed-phase column using UV detection at 262 nm. Urine was analysed directly after centrifugation to remove particulate matter. The detection limit was 0.5--1.0 micrograms/ml for serum and 5 micrograms/ml for urine. The method has been applied to the analyses of cefotaxime and desacetylcefotaxime in plasma, serum, urine, cerebrospinal fluid, saliva, and pus from infected wound secretions. Two additional metabolites, which are lactones in which the beta-lactam ring has been opened, could be separated by this method.     

Solid-phase extraction and determination of dansyl derivatives of unconjugated and acetylated polyamines by reversed-phase liquid chromatography: improved separation systems for polyamines in cerebrospinal fluid, urine and tissue

A sensitive and simple liquid chromatographic assay with fluorometric detection for unconjugated and acetylated polyamines in biological fluids is described. After precolumn derivatization with dansyl chloride, unconjugated polyamines and acetylated polyamines were extracted by elution from a Bond-Elut C18 column and then separated on a reversed-phase column with gradient elution. The complete analysis of unconjugated putrescine, spermidine, and spermine in either hydrolyzed urine, cerebrospinal fluid or tissue could be accomplished within 20-26 min, while the simultaneous analysis of unconjugated polyamines and monoacetylpolyamines could be completed within 40 min. Unhydrolyzed urine and cerebrospinal fluid required a Bond-Elut cation-exchange clean-up before dansylation. Standard curves for the assay were linear up to 20 nmol/ml, and the within-day and day-to-day coefficients of variation were between 1.1 and 4.6% and between 1.6 and 11.8%, respectively. Results obtained with the method were compared with results obtained with a well established modified amino acid analyzer method for urine, tissue and cerebrospinal fluid samples. The correlation coefficients between these two methods were in the range 0.933-0.996. Detection limits between 50 and 150 fmol were achieved for unconjugated and acetylated polyamines. Of more than twenty drugs and amines tested for possible interference with the assay, only normetanephrine was found to have the same retention time as the internal standard 1,6-diaminohexane.     

高效液相色谱-间接光度检测法测定体液中西梭霉素

采用高效液相色谱－间接光度检测法（ＨＰＬＣ－ＩＰＤ）,在流动相中加入具有紫外检测响应的检测剂烟酰胺,用紫外检测器直接测定体液中无紫外吸收的西梭霉素含量。固定相为ＳｐｈｅｒｉｓｏｒｂＣ１８,流动相为甲醇－乙腈－水（２０∶１０∶７０）,内含磷酸０．０５ｍｏｌ／Ｌ、烟酰胺０．５ｍｍｏｌ／Ｌ、庚烷磺酸钠５ｍｍｏｌ／Ｌ。血清样品中西梭霉素平均回收率为９６．９２％±４．６３％,日内和日间变异系数分别为４．７５％和５．６５％。     

Determination of phenolic compounds derived from hydrolysable tannins in biological matrices by RP-HPLC

An RP-HPLC method for the determination of four phenolic compounds: gallic acid (GA), pyrogallol (PY), resorcinol (RE) and ellagic acid (EA), derived from hydrolysable tannins is reported. Separation was achieved on a SunFire C18 (250 x 4.6 mm id, 5 microm) column at 40 degrees C with gradient elution. UV detection at 280 nm was applied. The developed method was validated in terms of linearity, accuracy and precision. Satisfactory repeatability and between day precision were noticed with RSD values lower than 3%. Recoveries from different biological samples ranged from 91.50 to 105.25%. The LODs were estimated as 1.70 mg/L for PY, 1.68 mg/L for GA, 1.52 mg/L for RE and 0.98 mg/L for EA with a 20 microL injection volume. The method was applied for the determination of these compounds in oak leaves and in ruminal fluid and urine samples taken from beef cattle fed with oak leaves. The proposed method could be used in ruminant nutrition studies to verify the effect that a diet rich in tannins have on ruminal fermentation and to determine the toxicity of these compounds.     

Simultaneous liquid chromatographic determination of seventeen of the major monoamine neurotransmitters, precursors and metabolites. II. Assessment of human brain and cerebrospinal fluid concentrations

The optimized chromatographic method procedure presented in Part I was employed for the assessment of human brain and cerebrospinal fluid neurotransmitters levels. The optimized sample preparation and chromatographic conditions permitted a rapid (less than 25 min), sensitive and semi-automated high-performance liquid chromatographic analysis which measures all major monoamine neurotransmitters, precursors and metabolites in human brain and cerebrospinal fluid. The brain specimen was deproteinized with perchloric acid (containing Na2EDTA and sodium sulphite), the internal standard and heparin were added and the samples were sonicated, centrifuged, filtered and injected directly into the chromatographic system. Cerebrospinal fluid was handled in a similar manner except that sonication was excluded. The regional distribution of monoamine neurotransmitter concentrations in human brain and cerebrospinal fluid is presented.     

Measurement of K-27, an oxime-type cholinesterase reactivator by high-performance liquid chromatography with electrochemical detection from different biological samples

K-27 is a bisquaternary asymmetric pyridinium aldoxime-type cholinesterase reactivator of use in the treatment of poisoning with organophosphorous esterase inhibitors. A sensitive, simple and reliable reverse-phase high-performance liquid chromatographic method with electrochemical detection was developed for the measurement of K-27 concentrations in rat brain, cerebrospinal fluid, serum and urine samples. Male Wistar rats were treated intramuscularly with K-27 and the samples were collected 60 min later. Separation was carried out on an octadecyl silica stationary phase and a disodium phosphate solution (pH 3.7) containing citric acid, octane sulphonic acid and acetonitrile served as mobile phase. Measurements were carried out at 30 degrees C at E(ox) 0.65 V. The calibration curve was linear through the range of 10-250 ng/mL. Accuracy, precision and the limit of detection calculated were satisfactory according to internationally accepted criteria. Limit of quantitation was 10 ng/mL. The method developed is reliable and sensitive enough for monitoring K-27 levels from different biological samples including as little as 10 microL of cerebrospinal fluid. The method--with slight modification in the composition of the mobile phase--can be used to measure a wide range of other related pyridinium aldoxime-type cholinesterase reactivators.     

High-performance liquid chromatographic determination of morphine, morphine-3-glucuronide, morphine-6-glucuronide and codeine in biological samples using multi-wavelength forward optical detection.

An isocratic high-performance liquid chromatographic method has been developed for the determination of morphine, morphine-3-glucuronide, morphine-6-glucuronide and codeine in plasma, urine and cerebrospinal fluid. The use of an efficient solid-phase extraction procedure together with a forward optical scanning detector allows a detection limit of 500 pg/ml. The method was evaluated by examination of biological samples taken from newborn infants following the intravenous administration of morphine sulfate.     

Automated high-throughput method using solid-phase microextraction-liquid chromatography-tandem mass spectrometry for the determination of ochratoxin A in human urine

A new automated, high-throughput method for the determination of ochratoxin A (OTA) in human urine samples has been optimized and validated using solid-phase microextraction coupled to liquid chromatography-tandem mass spectrometry (SPME-LC-MS/MS). High-throughput was achieved by simultaneous preparation of up to 96 samples using multi-fiber SPME device and multi-well plates. A carbon-tape coating was chosen for the first time as the best extracting phase for this contaminant. The proposed method required only minimal sample pre-treatment to adjust sample pH to 3.0 using a dilution (1:1) with 0.5M phosphate-buffered saline. A simple gradient guaranteed a good chromatographic separation from matrix interferences in only 8min. Relative recovery (%), precision and linearity validation results met Food and Drug Administration acceptance criteria at three concentration levels (1, 10, and 50ng/mL), indicating excellent performance of the proposed method. Limits of detection and quantitation were 0.3 and 0.7ng/mL in urine, respectively. OTA determination in urine is a good marker for human exposure to this mycotoxin. It is also less invasive than blood analysis. This method is fully automated and the SPME technique is simpler, less time-consuming and cheaper compared with most widely adopted clean-up procedures for OTA extraction from urine.     

Quantification of myo‐inositol, 1,5‐anhydro‐ D‐sorbitol,and D‐chiro‐inositol using high‐performance liquid chromatography with electrochemical detection in very small volume clinical samples

Inositol is a six‐carbon sugar alcohol and is one of nine biologically significant isomers of hexahydroxycyclohexane. Myo‐inositol is the primary biologically active form and is present in higher concentrations in the fetus and newborn than in adults. It is currently being examined for the prevention of retinopathy of prematurity in newborn preterm infants. A robust method for quantifying myo‐inositol (MI), D ‐chiro‐inositol (DCI) and 1,5‐anhydro‐ D ‐sorbitol (ADS) in very small‐volume (25 μL) urine, blood serum and/or plasma samples was developed. Using a multiple‐column, multiple mobile phase liquid chromatographic system with electrochemical detection, the method was validated with respect to (a) selectivity, (b) accuracy/recovery, (c) precision/reproducibility, (d) sensitivity, (e) stability and (f) ruggedness. The standard curve was linear and ranged from 0.5 to 30 mg/L for each of the three analytes. Above‐mentioned performance measures were within acceptable limits described in the Food and Drug Administration's Guidance for Industry: Bioanalytical Method Validation. The method was validated using blood serum and plasma collected using four common anticoagulants, and also by quantifying the accuracy and sensitivity of MI measured in simulated urine samples recovered from preterm infant diaper systems. The method performs satisfactorily measuring the three most common inositol isomers on 25 μL clinical samples of serum, plasma, milk, and/or urine. Similar performance is seen testing larger volume samples of infant formulas and infant formula ingredients. MI, ADS and DCI may be accurately tested in urine samples collected from five different preterm infant diapers if the urine volume is greater than 2–5 mL. Copyright © 2015 John Wiley & Sons, Ltd.     

Electrophoretic separation of tryptophan enantiomers in biological samples.

A method for the determination of D- and L-tryptophan (Trp) in biological samples is described. The amino acid enantiomers were precolumn-derivatized with a fluorescence tagging reagent, naphthalene-2,3-dialdehyde (NDA). In the presence of hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD) as the chiral selector, NDA-tagged Trp enantiomers were well resolved by micellar electrokinetic chromatography (MEKC). Using laser induced fluorescence (LIF) detection, a detection limit of 3.3 x 10(-8) M Trp was obtained. The method was applied to the determination of Trp enantiomers in biological samples including human urine and cerebrospinal fluid (CSF), rat brain tissue, and Aplysia ganglia. No interference from other amino acids or the endogenous compounds in the sample matrices was observed. D-Trp was found at the sub-microM level in human urine samples collected from several healthy subjects. Further, the determination of DL-Trp residues in small quantities (10 microg) of peptides after acid hydrolysis is demonstrated.     

Simultaneous Gas Chromatographic Determination of Diazapam and its Major Metabolites in Human Plasma,Urine, and Saliva

Abstract

A glc analysis method was developed for the simultaneous determination of diazepam (I), and its major metabolites N-desmethyldiazepam (II), oxazepam (III), and hydroxydiazepam (IV) in human plasma, urine, and saliva. Medazepam (V) was used as the internal standard to control extraction efficiency and permit precise and accurate determination of I-IV. Extraction and glc analysis of physiological fluid samples in triplicate required only 40 minutes using the developed method. Three human subjects were given either 20 or 5 mg of I via oral administration and serial specimens of blood, urine, and saliva collected. All samples were analyzed using the developed assay method. Results indicate that the method could be applied to pharmacokinetic studies of I where plasma, urine, and/or saliva is to be monitored.     

Stereoselective Determination of Ornidazole Enantiomers in Human Plasma and Urine Samples by Chiral LC: Application to Pharmacokinetic Study

A stereoselective liquid chromatographic method to determine the enantiomers of ornidazole in human plasma and urine has been developed and validated. After addition of the internal standard (naproxen), samples were acidified and extracted with diethyl ether. The separation was performed on a Chiralcel OB-H column, using hexane-ethanol- glacial acetic acid (94:6:0.08, v/v) as the mobile phase. The method was validated for specificity, linearity, sensitivity, precision, accuracy and stability. For each enantiomer of ornidazole, linear calibration curves were obtained over the concentration range of 0.16–20 μg mL^?1 in plasma and 0.32–20 μg mL^?1 in urine. For both enantiomers of ornidazole in plasma and urine, the coefficient of variation for precision were consistently less than 12% and accuracy were within ±14% in terms of relative error. Application of the method to a preliminary pharmacokinetic study showed that this validated method was qualified for the direct determination of ornidazole enantiomers in human plasma and urine.     

Combined quantification of paclitaxel,docetaxel and ritonavir in human feces and urine using LC‐MS/MS

A combined assay for the determination of paclitaxel, docetaxel and ritonavir in human feces and urine is described. The drugs were extracted from 200 μL urine or 50 mg feces followed by high‐performance liquid chromatography analysis coupled with positive ionization electrospray tandem mass spectrometry. The validation program included calibration model, accuracy and precision, carry‐over, dilution test, specificity and selectivity, matrix effect, recovery and stability. Acceptance criteria were according to US Food and Drug Administration guidelines on bioanalytical method validation. The validated range was 0.5–500 ng/mL for paclitaxel and docetaxel, 2–2000 ng/mL for ritonavir in urine, 2–2000 ng/mg for paclitaxel and docetaxel, and 8–8000 ng/mg for ritonavir in feces. Inter‐assay accuracy and precision were tested for all analytes at four concentration levels and were within 8.5% and <10.2%, respectively, in both matrices. Recovery at three concentration levels was between 77 and 94% in feces samples and between 69 and 85% in urine samples. Method development, including feces homogenization and spiking blank urine samples, are discussed. We demonstrated that each of the applied drugs could be quantified successfully in urine and feces using the described assay. The method was successfully applied for quantification of the analytes in feces and urine samples of patients. Copyright © 2013 John Wiley & Sons, Ltd.     

高效液相色谱法检测人尿中皮质激素水平

建立了高效液相色谱法分析测定尿中皮质激素的方法，该法可同时测定尿中醛固酮、可的松及皮质醇的水平，最低检出限为０１ｍｇ／Ｌ，线性范围０５～２０ｍｇ／Ｌ，回收率达９５％。方法简便、灵敏度较高，可作为常规手段推广应用。