Determination of bisphenol-A in water by semi-micro column high-performance liquid chromatography using 2-methoxy-4-(2-phthalimidinyl)phenylsulfonyl chloride as a fluorescent labeling reagent.

A highly sensitive method for the determination of bisphenol-A in water with semi-micro column high-performance liquid chromatography using 2-methoxy-4-(2-phthalimidinyl)phenylsulfonyl chloride as a fluorescent labeling reagent has been developed. The labeling reaction was carried out at 70 degrees C for 20 min in borate buffer (pH 9.5). The derivative eluted at 11.6 min on a reversed-phase column with methanol-water (78:22, v/v) at a flow-rate of 0.2 ml/min. The fluorescence was monitored at 308 nm for excitation and 410 nm for emission. The detection limit (S/N = 3) was 10 fmol per injection. The labeling yield was about 95%.     

High-performance liquid chromatographic determination of acetone in blood and urine in the clinical diagnostic laboratory.

A method for the determination of acetone in plasma or urine by high-performance liquid chromatography (HPLC) was developed. Plasma specimens are deproteinized with acetonitrile (1:1, v/v) 2,4-dinitrophenylhydrazine (DNPH) is added to the supernatant or to filtered urine samples, similarly treated with acetonitrile (2:1, v/v) to prevent crystallization of the synthesized phenylhydrazone. An aliquot (20 microliters) of the reaction mixture was subjected to HPLC at ambient temperature using a reversed-phase Pecosphere 3 x 3 C18 column with acetonitrile-water (45:55, v/v) as eluent at a flow-rate of 1 ml/min and detection at 365 nm. Hydroxyacetone and acetoacetate phenylhydrazone derivatives do not interfere. The identification of acetone by its retention time was confirmed by comparison with a laboratory-synthesized acetone DNPH derivative. The concentration of acetone, eluted within 3 min, was determined by the peak-height method. The detection limit was 0.034 mmol/l; the relative standard deviations were less than 5% within run (n = 20) and less than 10% between run (n = 20).     

Measurement of bumetanide in plasma and urine by high-performance liquid chromatography and application to bumetanide disposition.

A high-performance liquid chromatographic method for the measurement of bumetanide in plasma and urine is described. Following precipitation of proteins with acetonitrile, bumetanide was extracted from plasma or urine on a 1-ml bonded-phase C18 column and eluted with acetonitrile. Piretanide dissolved in methanol was used as the internal standard. A C18 Radial Pak column and fluorescence detection (excitation wavelength 228 nm; emission wavelength 418 nm) were used. The mobile phase consisted of methanol-water-glacial acetic acid (66:34:1, v/v) delivered isocratically at a flow-rate of 1.2 ml/min. The lower limit of detection for this method was 5 ng/ml using 0.2 ml of plasma or urine. Nafcillin, but not other semi-synthetic penicillins, was the only commonly used drug that interfered with this assay. No interference from endogenous compounds was detected. For plasma, the inter-assay coefficients of variation of the method were 7.6 and 4.4% for samples containing 10 and 250 ng/ml bumetanide, respectively. The inter-assay coefficients of variation for urine samples containing 10 and 2000 ng/ml were 8.1 and 5.7%, respectively. The calibration curve was linear over the range 5-2000 ng/ml.     

Determination of bupropion using liquid chromatography with fluorescence detection in pharmaceutical preparations, human plasma and human urine

A novel pre-column derivatization reversed-phase high-performance liquid chromatography with fluorescence detection is described for the determination of bupropion in pharmaceutical preparation, human plasma and human urine using mexiletine as internal standard. The proposed method is based on the reaction of 4-chloro-7-nitrobenzofurazan (NBD-Cl) with bupropion to produce a fluorescent derivative. The derivative formed is monitored on a C18 (150 mm × 4.6 mm i.d., 5 μm) column using a mobile phase consisting of methanol-water 75:25 (v/v), at a flow-rate of 1.2 mL/min and detected fluorimetrically at λ(ex) = 458 and λ(em) = 533 nm. The assay was linear over the concentration ranges of 5-500 and 10-500 ng/mL for plasma and urine, respectively. The limits of detection and quantification were calculated to be 0.24 and 0.72 ng/mL for plasma and urine, respectively (inter-day results). The recoveries obtained for plasma and urine were 97.12% ± 0.45 and 96.00% ± 0.45, respectively. The method presents good performance in terms of precision, accuracy, specificity, linearity, detection and quantification limits and robustness. The proposed method is applied to determine bupropion in commercially available tablets. The results were compared with an ultraviolet spectrophotometry method using t- and F-tests.     

Determination of glyoxylic acid in urine by liquid chromatography with fluorescence detection, using a novel derivatization procedure based on the Petasis reaction

The Petasis reaction is the multi-component reaction of a carbonyl compound, amine, and arylboronic acid to form an α-amino acid or a β-aminoalcohol. In this work, as the first analytical application of the Petasis reaction, a high-performance liquid chromatographic (HPLC) method with fluorescence detection was developed for determination of glyoxylic acid. The glyoxylic acid was derivatized with 1-pyreneboronic acid, as fluorescent arylboronic acid, in the presence of N-methylbutylamine, as amine, to give a fluorescent α-amino acid. HPLC separation of the fluorescent derivative was performed within 30 min on an octyl column eluted with a gradient prepared from acetonitrile and 50 mmol L(-1) acetate buffer (pH 4.0). The detection limit (S/N=3) for glyoxylic acid was 5.0 nmol L(-1) (20 fmol/injection). The method can be used to determine the concentration of glyoxylic acid in human urine without interference from biological components.     

A sensitive liquid chromatographic method for the analysis of isovaleric and valeric acids in urine as fluorescent derivatives

A simple and sensitive isocratic liquid chromatographic method was developed for the analysis of isovaleric and valeric acids in human urine as biomarkers in metabolic acidosis. The method is based on the derivatization of isovaleric and valeric acids with a fluorescent reagent 2-(2-naphthoxy)ethyl-2-(piperidino)ethanesulfonate for labeling the analytes with the naphthoxy fluorophore. The resulting fluorescent derivatives of isovaleric and valeric acids were separated on a phenyl-hexyl column, using a mixed solvent of methanol-water-tetrahydrofuran (55:31:14, v/v) as the mobile phase. The separated derivatives were monitored with a fluorimetric detector (excitation at 225 nm and emission at 360 nm). The linear range of the method for the determination of isovaleric acid or valeric acid derivative was over 0.2 approximately 8.0 microM. The detection limit (signal to noise ratio=3 with 10 microl injected) of isovaleric acid or valeric acid was about 0.04 microM. Application of the method to the analysis of isovaleric acid in the urine of a patient with isovaleric acidemia proved feasible.     

Simultaneous micro-determination of nicotinamide and its major metabolites, N1-methyl-2-pyridone-5-carboxamide and N1-methyl-4-pyridone-3-carboxamide, by high-performance liquid chromatography

A simultaneous micro-determination of nicotinamide and its major metabolites, N1-methyl-2-pyridone-5-carboxamide (2-py) and N1-methyl-4-pyridone-3-carboxamide (4-py) by high-performance liquid chromatography is described. The method employs a 7-ODS-L (250 mm X 4.6 mm I.D., particle size 7 microns) column eluted with 10 mM potassium dihydrogenphosphate-acetonitrile (96:4, v/v; pH adjusted to 3.0 by the addition of concentrated phosphoric acid) at a flow-rate of 1.0 ml/min. The UV detector was set at 260 nm. The detection limits for nicotinamide, 2-py and 4-py were 10 pmol (1.22 ng), 2 pmol (304 pg) and 2 pmol (304 pg), respectively, at a signal-to-noise ratio 5:1. Isonicotinamide was used as an internal standard. The technique was applied to the analysis of rat and human urines. The total analysis time was ca. 15 min.     

Rapid assay for hard tissue collagen cross-links using isocratic ion-pair reversed-phase liquid chromatography

Following a detailed study, a rapid and sensitive assay for the naturally fluorescent collagen cross-links pyridinoline and deoxypyridinoline has been developed using ion-pair reversed-phase high-performance liquid chromatography in the presence of 1-octanesulphonic acid (OSA). Pyridinoline and deoxypyridinoline were separated on an Exsil 100 ODS, 5-microns column (100 mm X 4.6 mm I.D.) using 25 mM sodium formate, 5 mM OSA and 1 mM ethylenediaminetetraacetic acid adjusted to pH 3.25, containing 20% (v/v) methanol. The mobile phase flow-rate was 1.5 ml/min. Compounds were detected by their natural fluorescence (xenon lamp; excitation wavelength 290 nm, emission wavelength 400 nm). Peak areas were linear to 25 pmol injected for pyridinoline and 20 pmol injected for deoxypyridinoline (r = 0.99). Intra-assay coefficients of variation for urinary extracts were 7.65 and 9.07% (n = 10), respectively. Limit of detection (signal-to-noise ratio = 5) was 200 fmol injected. Quantification of the cross-links in acid hydrolysates and human urine samples was possible in under 15 min.     

在线固相萃取-液相色谱法直接测定水中超痕量多环芳烃

建立了在线固相萃取-液相色谱直接测定水体中16种超痕量多环芳烃（PAHs）的方法。水样经高速离心后，加入适量甲醇，配制成40%（体积分数）甲醇水溶液，直接进样2 mL至在线固相萃取流路，进行萃取富集，再通过阀切换将洗脱的PAHs转移至分析流路进行分离检测。16种PAHs在各自范围内线性关系良好，相关系数均大于0.996；方法的检出限为0.14~12.50 ng/L，其中苯并[a]芘（B（a）P）的检出限为0.38 ng/L。实际水样在10、40和200 ng/L加标水平下的加标回收率为76.1%~134.9%，RSD为0.3%~16.6%。B（a）P在1 ng/L加标水平下的回收率为71.8%~92.7%，RSD为3.9%。结果表明，该方法操作简单，灵敏度高，溶剂消耗量少，可满足水样中PAHs，尤其是B（a）P的超痕量分析要求。     

Determination of urinary free cortisol by high performance liquid chromatography with sulphuric acid-ethanol derivatization and column switching.

A rapid and highly sensitive determination method for urinary free cortisol has been developed using reversed phase high performance liquid chromatography (HPLC) with a precolumn for sulphuric acid-ethanol fluorescence derivatization and column switching. Urinary cortisol, eluted from the octadecylsilane-bonded silica (ODS) minicolumn with 90% aqueous ethanol, was derivatized with the addition of sulphuric acid only at ambient temperature. Cortisol derivatives injected directly onto the ODS precolumn were purified on-line. After switching the columns, the cortisol derivative was separated on an ODS analytical column with a retention time of 15.3 min and monitored at an emission wavelength of 520 nm (exitation wavelength of 365 nm) to decrease the detection limit to 0.26 microgram/dL (signal-to-noise ratio = 3). The automated HPLC operation resulted in good reproducibility and recovery of the stable cortisol derivative at 5 degrees C.     

Determination of exifone in human plasma and urine by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method with electrochemical detection was developed for the determination of exifone in human plasma and urine. Exifone was extracted from acidified plasma or neutralized urine with diethyl ether and the evaporated extracts were analysed on a C18 reversed-phase column. The compound was eluted in about 8 min with acetonitrile-0.3 M orthophosphoric acid (15:85, v/v) at a flow-rate of 0.9 ml/min. This method gave accurate and reproducible results; the calibration graphs were linear (r greater than 0.99) over the range of 2.8-360 nmol/l for plasma and 0.18-36 mumol/l for urine, and concentrations as low as 1 nmol/l in plasma could be quantified. These results allowed this assay to be used for determinations in single-dose pharmacokinetic studies.     

手性荧光衍生化反相高效液相法分离肾上腺素对映体

 以R (- ) /S (+) 4 (N ,N dimethylaminosulfonyl) 7 (3 iso thiocyanatopyrrolidino) 2 ,1,3 benzoxadiazole(DBD PyNCS)为手性荧光衍生化试剂 ,用反相高效液相法 (RP HPLC)对 DL 肾上腺素对映体进行分离。衍生化反应是在含有 7%吡啶的溶剂中 ,6 5℃温度条件下反应 35min ,生成具有荧光特性的肾上腺素非对映体衍生物(λex=4 6 0nm ,λem=5 5 0nm)。生成的非对映体衍生物在流动相为乙腈 水 (体积比为 2 8∶72 ) ,流速为 1 0mL/min时 ,在DiamonsilTMC18柱 (15 0mm× 4 6mmi d ,5 μm)上分离度可达 2 6。     

A sensitive and selective determination method of histamine by HPLC with intramolecular excimer-forming derivatization and fluorescence detection

A highly sensitive, selective and simple method is described for the determination of histamine by high-performance liquid chromatography (HPLC) with fluorescence detection. The method is based on an intramolecular excimer-forming fluorescence derivatization of histamine with 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase HPLC. Histamine, having two amino moieties in a molecule, was converted to the dipyrene-labeled derivative by reaction with PSE. The derivative afforded intramolecular excimer fluorescence (450-540 nm), which can clearly be discriminated from the monomer fluorescence (370-420 nm) emitted from PSE. Typically, a 10 micro L sample solution was mixed with 100 micro L of derivatization reagent solution, which was a mixture of 0.5 mm PSE in acetonitrile and 0.5 mm potassium carbonate in water (8:2, v/v). The derivatization was carried out at 100 degrees C for 90 min. The PSE derivative of histamine could be separated by reversed-phase ODS column with isocratic elution using acetonitrile:water (82:18, v/v) containing 0.03% triethylamine. The detection limit (singnal-to-noise ratio = 3) of histamine was 0.5 fmol for a 30 micro L injection. The method was successfully applied to the determination of histamine in human urine, and had enough selectivity and sensitivity for urinary histamine quantification.     

Determination of antitubercular drugs in urine and pharmaceuticals by LC using a gradient flow combined with programmed diode array photometric detection

The simultaneous determination of the antitubercular drugs rifampicin, pyrazinamide, isoniazid and the acetylisoniazid metabolite has been accomplished by LC, using a C-18 analytical column. The assayed drugs are usually administered together in the treatment of tuberculosis. Creatinine was also included in the chromatographic determination, in order to establish the curve of excretion of the drugs in urine. The chromatographic method uses a gradient flow in three steps, in conjunction with a programmed diode array photometric detection. In a 0.02 M potassium dihydrogen phosphate pH 7.0 buffer, a 5% (v/v) content of methanol for 1 min, a 8% (v/v) content of methanol for 3.4 min, and a 75% (v/v) content of methanol for 4 min were used. At 4.5 min, the wavelength value of detection was changed from 254 to 475 nm. Creatinine, acetylisoniazid, isoniazid and pyrazinamide were eluted in the first 4.5 min and rifampicin before 8 min. The method has been satisfactorily applied to the determination of the drugs in urine samples and in pharmaceuticals. The proposed LC method is simple, and a short time, less than 8 min is necessary for compounds elution.     

Ion-paired high-performance liquid chromatographic determination of mitoxantrone in physiological fluids

Mitoxantrone is one of the newer anthracenedione derivatives which has already been studied in phase I and II trials, where it has shown significant antitumor activity against a variety of human tumours. To determine the prolonged terminal half-life of mitoxantrone, we developed a sensitive high-performance liquid chromatographic method, providing a detection limit of 1 ng/ml of extracted serum. This system uses a C18 reversed-phase column. The mobile phase consists of a mixture of acetonitrile (30%, v/v) and an ammonium formate buffer (70%, v/v) with a pH of 2.7. Hexane sulphonic acid is added as an ion-pair former. Detection at a wavelength of 658 nm provides a highly selective system, showing no interfering peaks. Ametantrone, another anthracenedione derivative, is used as an internal standard. The extraction procedure for serum also uses hexane sulphonic acid in an ion-paired system. Because of the highly selective detection wavelength, urine samples can be injected without a sample clean-up procedure. This very sensitive method, combined with high selectivity and a fast and inexpensive serum clean-up procedure, has allowed us to document the prolonged terminal plasma half-life of mitoxantrone (levels of 2-5 ng/ml of plasma can still be detected six days after an intravenous infusion of 15 mg/m2 over 30 min). In urine an as yet unidentified metabolite was discovered.     

High-performance liquid chromatography assay for 1-deoxy-D-xylulose 5-phosphate synthase activity using fluorescence detection

A high-performance liquid chromatography assay for activity of 1-deoxy-D-xylulose 5-phosphate synthase, an early enzyme in the recently discovered 2-C-methyl-D-erythritol-4-phosphate pathway, was developed. In this assay, the enzymatic product 1-deoxy-D-xylulose was first derivatized with a fluorescent reagent 2-anthranilic acid, followed by separation using HPLC on a Nova-Pak phenyl column with a mobile phase containing CH3CN-water-1-butylamine-tetrahydrofuran-H3PO4 (2:97:0.125:0.5:0.25, v/v). The eluate was monitored by fluorescence detection at an excitation wavelength of 320 nm and an emission wavelength of 425 nm for quantitation of the fluorescent derivative. A linear response was obtained between 5 and 200 ng of 1-deoxy-D-xylulose. This assay was successfully applied to measure the 1-deoxy-D-xylulose 5-phosphate synthase activity in a recombinant E. coli overexpressing dxs gene. It demonstrated that this assay is simple, sensitive and selective compared to the methods used at present.     

Preparation of a fluorescent derivative of benzoylecgonine, and preliminary studies of its application to the analysis of urine.

A sensitive high-performance liquid chromatographic method using 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone (Br-DMEQ) as a fluorescent labeling reagent is described for the determination of benzoylecgonine (BE) and ecgonine (EC). The Br-DMEQ derivatives of BE and EC were separated on a C18 column and detected at 455 nm with excitation at 370 nm. The detection limits of the proposed method were 18.7 fmol for BE and 12.5 pmol for EC at a signal-to-noise ratio of 3. Relative standard deviations of five replicate measurements were 1.94% (10 pmol) and 2.98% (50 pmol) for BE and 6.3% (250 pmol) and 5.62% (1.25 pmol) for EC. This method was applied to the determination of BE in human urine. BE was extracted from urine by solvent extraction with chloroform-isopropyl alcohol (9:1, v/v) solution. Levels of 2.5.10(-8) M BE in urine (25 pmol/ml) could be determined.     

UV light-emitting diode-induced fluorescence detection combined with online sample concentration techniques for capillary electrophoresis.

The application of an ultraviolet (UV) light-emitting diode (LED) to on-line sample concentration/fluorescence detection in capillary electrophoresis (CE) is described. The utility of a UV-LED (peak emission wavelength at 380 nm, approximately 2 mW) for fluorescence detection was demonstrated by examining both a naturally fluorescent (riboflavin) compound and a nonfluorescent compound (tryptophan), respectively. The detection limit for riboflavin was determined to be 0.2 ppm by the normal MEKC mode, which was improved to 3-7 ppb when dynamic pH-junction technique was applied. On the other hand, the detection limit of the tryptophan derivative was determined to be 1.5 ppm using the MEKC mode, which was improved to 3 ppb when the sweeping-MEKC mode was applied. In an analysis of an actual sample, the concentrations of riboflavin in beer, and tryptophan in urine and milk samples were determined, respectively.     

Quantification of glutathione in plasma samples by HPLC using 4-fluoro-7-nitrobenzofurazan as a fluorescent labeling reagent

A rapid and highly sensitive high-performance liquid chromatograpy method with fluorescence detection has been developed for determination of glutathione (GSH) in human plasma. A simple pre-column derivatization procedure with 7-flouro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) reagent was employed. The separation of the derivatized glutathione was performed using a mobile phase consisting of phosphate buffer (0.02 mol/L, pH 6.0)-acetonitrile (77:23, v/v) at a flow rate of 1.0 mL/min with the column temperature 2°C. The eluted derivatives were fluorometrically detected at an excitation wavelength 470 nm and an emission wavelength 530 nm. Under the optimum chromatographic conditions, the calibration curve was linear over the range of 0.1 μmol/L to 10.0 μmol/L with the correlation coefficient of 0.9988. The precision of the method was satisfactory with the intra- and inter-day coefficient of variation being 6.3%, 6.9%, respectively. This method has been used to determine glutathione concentrations in plasma samples from healthy individuals.     

高效液相色谱法同时测定尿中新喋呤和生物喋呤

建立了同时分离检测尿中新喋呤（neopterin,NP）和生物喋呤(biopterin,BP)的高效液相色谱（HPLC）-荧光检测方法。采用Hypersil BDS C18柱、甲醇-水(体积比为10∶90)流动相(流速0.5 mL/min)、激发波长360 nm、发射波长 440 nm、柱温20 ℃的色谱条件,同时分离测定了尿中的NP和BP。尿标本经三氯乙酸处理,在4 ℃下,以12000 r/min的速率离心15 min,上清液用碱中和后,取30　μL直接进样。研究结果表明,NP的线性范围为0.12～10