Determination of fluoxetine and norfluoxetine in rat plasma by HPLC with pre-column derivatization and fluorescence detection

Both fluoxetine (FLX) and its N-demethylated metabolite, norfluoxetine (NFLX), have been reported to be potent serotonin-reuptake inhibitors. A sensitive and reliable method that allows simultaneous quantification of their plasma levels would be valuable and was developed in this work. The procedure included extraction of FLX and NFLX from plasma, fluorescence derivatization with 4-(N-chloroformylmethyl-N-methyl) amino-7-nitro-2,1,3-benzoxadiazole (NBD-COCl), separation of the derivatives on an octadecylsilica column with acetonitrile-water (55:45,v/v) as mobile phase and fluorescence detection with emission at 537 nm and excitation at 478 nm. The calibration curves were linear for FLX and NFLX concentration over the range of 10-1000 nM (r = 0.9992 and r = 0.9997) and the limits of quantitation were 10 nM in 100 micro L of plasma. Precision of intra- and inter-day RSD of less than 12% and accuracy of intra- and inter-day RE within -6.0-13% were achieved. The method described was applied to analysis of the plasma samples from rats treated with FLX hydrochloride and to the pharmacokinetic study.     

Simple HPLC determination of colistin in medicated feeds by pre-column derivatization and fluorescence detection

Summary A simple and sensitive HPLC method was developed for the determination of colistin antibiotic in feeds employing pre-column derivatization and fluorescence detection. Extraction of colistin in feeds was by sonication and shaking with 0.1 M HCl. Pre-column derivatization was with phthaldialdehyde and 2-mercaptoethanol (ME) in borate buffer (pH 10.5) to obtain a fluorescent derivative. Elution of the derivative onto an Ultracarb 5 μm ODS column was by using acetonitrile—ultrapure water (75∶25). Detection was by spectrofluorimetry at 340 nm (excitation wavelength) and 440 nm (emission wavelength). Total elution time was <20 min. The applicability of the validated method was tested by analyzing commercial medicated feeds without any interference from the matrix.     

Determination of the impurity of isopentylamine hydrochloride in the intermediate of bortezomib by HPLC with pre-column derivatizationEI北大核心CSCD

A method was developed for the simultaneous determination of （R）-pinacol-1-amino-3-methylbutylborate hydrochloride and isopentylamine hydrochloride by pre-column derivatization high performance liquid chromatography （HPLC） with dichloromethane as solvent,triethylamine as acid binding agent and benzoyl chloride as derivative reagent. （R）-pinacol-1-amino-3-methylbutylborate hydrochloride and isopentylamine hydrochloride reacted with derivatization reagent in an ice bath for 10 min. The derivatives were qualitatively determined by HPLC-mass spectrometry（HPLC-MS）. HPLC was performed on an AgelaVenusil MP C18 （2）column using methanol-water （70∶30,V/V）as mobile phase. The flow rate was 1.0 mL/min,the detection wavelength was 236 nm and the column temperature was 35℃ . The UV absorption produced after derivatization,which could be detected by HPLC with good specificity. Isopentylamine hydrochloride derivative showed an excellent linearity in the range of 0.075-30 μµg/mL,with the limit of quantification and the limit of detection（LOD）of 0.075 and 0.030 μµg/mL,respectively. And the S/N of the LOQ and the LOD were 44.6 and 7.8,respectively. The average recoveries were 97.7%-104.0% with the RSD of 2.9%. The method is suitable for the determination of isoamylamine hydrochloride and has significant reference value for the determination of other amine salt without UV absorption. © 2022, Youke Publishing Co.,Ltd. All rights reserved.     

Determination of fluvoxamine in rat plasma by HPLC with pre-column derivatization and fluorescence detection using 4-fluoro-7-nitro-2,1,3-benzoxadiazole

A sensitive, simple and reliable method using high-performance liquid chromatographic (HPLC) assay of fluvoxamine (FLU), a selective serotonin reuptake inhibitor (SSRI), in rat plasma after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed in this study. Extracted plasma samples were mixed with NBD-F at 60 degrees C for 5 min and injected into HPLC. Retention times of FLU and an internal standard (propafenone) derivative were 15.5 and 13.5 min, respectively. The calibration curve was linear over the range 0.015-1.5 microg/mL (r2 = 0.9985) and the lower limits of detection and quantification of FLU were 0.008 and 0.015 microg/mL, respectively, in 100 microL of plasma. The derivative sample was stable at 4 degrees C for 1 day. The coefficients of variation for intra-day and inter-day assay of FLU were less than 8.3 and 9.6%, respectively. Other SSRIs and centrally acting drugs did not interfere with the peak of the FLU derivative. The method was applied for analysis of the plasma samples from rats treated with FLU. These results indicate that the method presented is useful to determine the FLU levels in rat plasma of volumes as small as 100 microL and can be applied to pharmacokinetic studies.     

Determination of sulfonamide residues in cultured sea cucumber by pre-column derivatization capillary electrophoresis with fluorescence detection

A simple and mild method for the separation of sulfonamide residues based on a condensation reaction with O-phthalaldehyde solution (OPA) as labeling reagent with capillary electrophoresis has been developed. A 58.5 cm × 50 μm i.d. (50 cm effective length) untreated fused-silica capillary was used. To optimize the separation conditions, the background electrolyte concentration, pH, column temperature, voltage and other factors were evaluated. The optimal separation conditions were as follows: 20 mmol L^?1 borate buffer; pH 9.1; column temperature 20 °C; separation voltage 18 kV, pressure 50 mbar and injection time 8 s. Under the optimal conditions, 10 kinds of sulfonamide derivatives could be well-separated within 8 min, and the linear ranges were 0.35–100 μg kg^?1. The detection limit (at a signal-to-noise ratio of 3) was in the range of 0.12–0.25 μg kg^?1, and the quantification limit (at a signal-to-noise ratio of 10) was in the range of 0.35–0.70 μg kg^?1. The sulfonamide residues from cultured sea cucumber samples were determined under the optimal conditions with satisfactory results.     

Determination of acrylamide in starch-based foods by HPLC with pre-column ultraviolet derivatization

A new method is developed for the determination of acrylamide in starch-based foods. The method included the extraction of acrylamide with water, defatting with hexane, derivatization with potassium bromate (KBrO(3)) and potassium bromide (KBr), liquid-liquid extraction with ethyl acetate-hexane (4:1), and concentration. The final analyte (2-bromopropenamide, 2-BPA) is analyzed by high-performance liquid chromatography coupled with diode array detection for quantification and by gas chromatography coupled to mass spectrometry for confirmation. The chromatographic analysis is performed on an ODS-3 C(18) column, and good retention and peak response of acrylamide are achieved under the optimal conditions. The limit of detection and quantitation are estimated to be 15 and 50 μg/kg, respectively. The recoveries of acrylamide from the commercial samples are spiked at levels of 50-1000 μg/kg, and range between 89.6 and 102.0%. These results show that this method should be regarded as a new, low-cost, and robust alternative for conventional investigation of acrylamide.     

柱前衍生高效液相色谱法测定尼莫地平注射液中的甲醛和乙醛

建立柱前衍生高效液相色谱测定尼莫地平注射液中甲醛和乙醛含量的方法.以2,4-二硝基苯肼为衍生化试剂,室温反应30 min,采用高效液相色谱法检测,色谱柱为Waters XBridge C8柱(250 mm×4.6 mm,5μm),流动相为乙腈–水(体积比为50:50),等度洗脱,流量为1.0 mL/min,柱温为30℃...     

Determination of optical impurity of pregabalin by HPLC with pre-column chiral derivatization

A new method for determining the optical impurity of Pregabalin is developed. The method is based on Pregabalin, and its isomers can be derivatized with Na-5-fluoro-2,4-dinitrophenyl-5-L-alanine amide. These derivated compounds can be separated by an ordinary chromatography column (Inertsil ODS-2.5 microm, 250 mmx4.6 mm i.d.). Phosphoric acid buffer and acetonitrile (55:45, v/v) are used as mobile phase and 1.0 mL/min flow rate at room temperature. The detective wavelength is fixed at 340 nm. The results indicate that the limit of detection of R model optical impurity 1.1x10(-8) g/mL (signal-to-noise=3), accuracy, and repeatability is satisfied. Therefore, the method can be used for the quality control of Pregabalin.     

高效液相色谱法测定盐酸甲氯芬酯胶囊的含量

建立了用高效液相色谱测定盐酸甲氯芬酯胶囊含量的方法.采用Hypersil C18柱(5 μm,4.6 mm i.d.×200 mm),流动相为V(乙腈)∶V(0.12% NH4HCO3-0.50%三乙胺)溶液＝33∶67 (甲基磺酸调pH至3.0),流速: 1.0 mL/min,检测波长为225 nm.盐酸甲氯芬酯的线性范围为1.632～163.2 μg/mL,平均回收率为99.67%,RSD=1.8% (n=9).     

柱前衍生反相高效液相色谱-荧光检测法测定大鼠血浆中的奈替米星

建立了一种简便、灵敏的氯甲酸芴甲酯(FMOC-Cl)柱前衍生反相高效液相色谱-荧光检测血浆中奈替米星的新方法,同时研究了其药代动力学。对色谱条件进行了优化,采用ZORBAX Eclipse XDB-C8柱(150 mm×4.6 mm,5 μm),流动相为乙腈-水(体积比为85:15),流速为1.0 mL/min,荧光检测激发波长为265 nm,发射波长为315 nm,得到奈替米星的平均加标回收率为96.62%～100.84%(n=3),对奈替米星检测的线性范围为0.045～8.88 mg/L,相关系数为0.9993,方法的日内与日间精密度分别低于3%与3.5%,最低检出限(S/N=3)与定量限(以3倍检出限计)分别为0.01和0.03 mg/L。方法简便、快速、灵敏,样品用量少(30 μL奈替米星血浆溶液已能满足该药含量的测定以及药物代谢的研究),为大鼠体内奈替米星的药代动力学研究提供了可靠的分析手段。     

Simultaneous determination of amantadine and rimantadine by HPLC in rat plasma with pre-column derivatization and fluorescence detection for pharmacokinetic studies

We investigated simultaneous high-performance liquid chromatographic (HPLC) determination of amantadine hydrochloride (AMA) and rimantadine hydrochloride (RIM) levels in rat plasma after fluorescent derivatization with o-phthalaldehyde and 2-mercaptoethanol. Afterwards, the method was applied to determine their pharmacokinetics. The retention times of AMA and RIM derivatives were 12.6 and 22.2 min and the lower limits of detection were 0.025 and 0.016 microg/mL, respectively. The coefficients of variation for intra- and inter-day assay of AMA and RIM were less than 5.1 and 7.6%, respectively. After i.v. administration of AMA or RIM to rats, the total body clearance and distribution volume at the steady-state of RIM were higher than those of AMA. Bioavailability of AMA and RIM was 34.9 and 37.2%, respectively. When AMA and RIM were p.o. co-administered, the area under the plasma concentration--time curve of RIM was significantly lower than that after RIM alone. On the other hand, pharmacokinetic parameters of AMA did not significantly change. These results indicate that our HPLC assay is simple, rapid, sensitive and reproducible for simultaneously determining AMA and RIM concentrations in rat plasma and is applicable to their pharmacokinetic studies. Also, co-administration of AMA and RIM may result in the lack of pharmacological effects of RIM.     

Determination of histamine and histidine by capillary zone electrophoresis with pre-column naphthalene-2,3-dicarboxaldehyde derivatization and fluorescence detection

A rapid and sensitive method was developed for the simultaneous determination of histamine and histidine by capillary zone electrophoresis with lamp-induced fluorescence detection. A fluoregenic derivatization reagent, naphthalene-2,3-dicarboxaldehyde (NDA) was successfully applied to label the histamine and histidine respectively. The derivatization conditions and separation parameters including pH and concentration of electrolyte and sample injection were optimized in detail. The optimal derivatization reaction was performed with 1.0 mM NDA, 20 mM NaCN, and 20 mM borate buffer, pH 9.1 for 15 min. The separation of NDA-tagged histamine and histidine could be achieved in less than 200 s with 40 mM phosphate buffer (pH 5.8) as the running buffer. The detection limits for histamine and histidine were 5.5 x 10(-9) and 3.8 x 10(-9) M, respectively (S/N = 3). The relative standard derivations for migration time and peak height of derivatives were less than 1.5 and 5.0%, respectively. The method was successfully applied to the analysis of histamine and histidine in the P815 mastocytoma cells and the beer samples.     

Determination of memantine in plasma and vitreous humour by HPLC with precolumn derivatization and fluorescence detection

A new HPLC procedure with precolumn derivatization and rimantadine as the internal standard for determining memantine, a candidate agent for the treatment of glaucoma in plasma and vitreous humour, has been developed and validated. Precolumn derivatization was performed with 9-fluorenylmethyl-chloroformate-chloride (FMOC-Cl) as the derivatization reagent and followed by a liquid-liquid extraction with n-hexane. Optimal conditions for derivatization were an FMOC-Cl concentration of 1.5 mM, a reaction time of 20 min, the temperature at 30°C, the borate buffer pH 8.5, and a borate buffer-acetonitrile ratio of 1:1. The derivatives were analyzed by isocratic HPLC with the fluorescence detector λex 260 nm λem 315 nm on a Novapack C(18) reversed-phase column with a mobile phase of acetonitrile-water (73:27, v/v), 40°C, and a flow rate of 1.2 mL/min. The linear range was 10-1000 ng/mL with a quantification limit of ～ 10 ng/mL for both types of samples. This analytical method may be suitable for using in ocular availability studies.     

Determination of paracetamol (acetaminophen) by HPLC with post-column enzymatic derivatization and fluorescence detection

Summary A novel method is described for the determination of paracetamol (acetaminophen;N-acetyl-4-aminophenol) in urine. After reversed-phase HPLC separation, paracetamol is oxidized by H₂O₂ with horseradish peroxidase catalysis. Detection is performed fluorimetrically at an excitation wavelength of 329 nm and an emission wavelength of 435 nm. Urine samples were spiked with paracetamol, diluted, and injected directly without further pretreatment. Under these conditions, the limit of detection was 2×10⁻⁸ molL⁻¹, and the limit of quantification was 7×10⁻⁸ molL⁻¹. The method was validated by two different approaches based on HPLC with UV-Vis detection.     

Determination of catecholamines as their N-hydroxy-succinimidyl-3-indolylacetate derivatives by pre-column derivatization HPLC separation and fluorescent detection

N-Hydroxysuccinimidyl-3-indolylacetate (SIIA) is a new fluorescent derivatizing reagent with an indole ring and ?an N-hydroxysuccinimide ester functionality. It can react with catecholamines under mild conditions to form corresponding amides, which have strong fluorescence at λ_ex/λ_em = 301 nm/?365 nm. This paper covers the RP-HPLC separation and fluorescent determination of derivatized catecholamines with SIIA. In a mobile phase of methanol-water (36/64, v/v) containing H₃cit-Na₂HPO₄ buffer (pH = 4.00, 10 mmol/L), the derivatives of norepinephrine (NE), epinephrine (E) and dopamine (DA) were eluted within 15 min on a C₁₈ column. The detection limits were 0.043, 0.13 and 0.18 pmol, respectively, when the ratio of signal to noise (S/N) was 3. The excessive reagent is rapidly hydrolyzed to 3-indolylacetic acid (IA) that can be easily separated from derivatives. Received: 16 April 1999 / Revised: 13 July 1999 / /Accepted: 15 July 1999     

Determination of catecholamines as their N-hydroxy-succinimidyl-3-indolylacetate derivatives by pre-column derivatization HPLC separation and fluorescent detection

N-Hydroxysuccinimidyl-3-indolylacetate (SIIA) is a new fluorescent derivatizing reagent with an indole ring and ¶an N-hydroxysuccinimide ester functionality. It can react with catecholamines under mild conditions to form corresponding amides, which have strong fluorescence at λ_ex/λ_em = 301 nm/¶365 nm. This paper covers the RP-HPLC separation and fluorescent determination of derivatized catecholamines with SIIA. In a mobile phase of methanol-water (36/64, v/v) containing H₃cit-Na₂HPO₄ buffer (pH = 4.00, 10 mmol/L), the derivatives of norepinephrine (NE), epinephrine (E) and dopamine (DA) were eluted within 15 min on a C₁₈ column. The detection limits were 0.043, 0.13 and 0.18 pmol, respectively, when the ratio of signal to noise (S/N) was 3. The excessive reagent is rapidly hydrolyzed to 3-indolylacetic acid (IA) that can be easily separated from derivatives.     

Determination of kynurenine levels in rat plasma by high-performance liquid chromatography with pre-column fluorescence derivatization

Kynurenine (KYN), a tryptophan metabolite, is a crucial compound for modulating neurotransmission because it can be metabolized in vivo into both quinolinic acid and kynurenic acid, which are the agonist and antagonist, respectively, of N-methyl-d-aspartate receptor. For the highly sensitive detection of KYN by high-performance liquid chromatography (HPLC), a fluorescence derivatization of KYN with a benzofurazan-type fluorogenic reagent, 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was investigated in the present study. KYN was derivatized with DBD-F (DBD-KYN) at 60 °C for 30 min, and separated on an octadecylsilica column with a gradient elution of the mobile phase, which consists of 0.1% formic acid in acetonitrile/methanol/water. DBD-KYN was detected fluorimetrically at 553 nm with an excitation wavelength of 431 nm. The limits of detection and quantification were approximately 0.30 pmol [signal-to-noise ratio (S/N) 3] and 1.0 pmol (S/N, 10) on column, respectively. Plasma KYN levels were successfully determined using 10 μL of rat plasma with satisfactory precision and accuracy. Intra- and inter-day precisions and accuracies were 1.7-6.8%, and −10 to 9.6%, respectively. KYN levels in plasma of male Sprague-Dawley rats (7 weeks old) were approximately 2.4 ± 0.32 μmol L⁻¹ (n = 4). The proposed HPLC method was applied to determine KYN levels in the plasma of ketamine-treated rats—the animal model of schizophrenia.