High Performance Liquid Chromatography With Fluorescence Detection Method Research Articles

Development and Validation of 96-Microwell-Based Spectrophotometric and High-Performance Liquid Chromatography with Fluorescence Detection Methods with High Throughput for Quantitation of Duvelisib and Seliciclib in Their Bulk Forms and Capsules

The Food and Drug Administration (FDA) has approved duvelisib (DUV) for managing follicular lymphoma, small lymphocytic lymphoma, and relapsed or refractory chronic lymphocytic leukemia. Seliciclib (SEL) is a candidate drug for these cancers, neurodegenerative disorders, renal diseases, several viral infections, and chronic inflammation disorders. This work describes the development and validation of a 96-microwell-based spectrophotometric method (MW-SPM) and a high-performance liquid chromatography with fluorescence detection method (HPLC-FD) for the quantitation of DUV and SEL in their bulk forms and capsules. The MW-SPM is based on the formation of colored charge transfer complexes (CTCs) as products for the reactions of DUV and SEL, as n-electron donors, with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), as a π-electron acceptor. The absorption intensity of the CTCs was measured by using an absorbance plate reader at 450 nm. The stoichiometric ratios of DUV:DDQ and SEL:DDQ were 1:1 and 1:2, respectively, and accordingly the reaction mechanisms were postulated. The HPLC-FD involved the chromatographic separation of DUV and SEL on a Hypersil™ Phenyl HPLC column (250 mm length × 4.6 mm i.d., 5 μm particle diameter) with a mobile phase composed of acetonitrile:acetate buffer, pH 4.5 (35:65, v/v) at a flow rate of 2.2 mL/min. DUV and SEL were detected at 370 nm after excitation at 280 nm. SEL was used as an internal standard (IS) for quantitation of DUV, and DUV was used as an IS for quantitation of SEL. Both MW-SPM and HPLC-FD were validated according to the guidelines of the International Council for Harmonization (ICH) for validation of analytical procedures. The linear ranges for both DUV and SEL were 14.52–200 µg/well (100 µL) and 0.12–3.2 µg/mL for MW-SPM and HPLC-FD, respectively. LOD values in MW-SPM for DUV and SEL were 4.4 and 3.17 µg/well, respectively; however, those for HPLC-FD were 0.03 and 0.05 µg/mL, respectively. The accuracy and precision of both methods were confirmed as the recovery values were ≥98.5% and the values of relative standard deviations (RSD) were ≤2.41%. Both methods were satisfactorily applied to the quantitation of DUV and SEL in their capsules; the mean recovery values were ≥99.2%. Both methods have simple procedures and high analytical throughput. Moreover, they consume a small volume of organic solvent; thus, they are economic and eco-friendly. Accordingly, the methods are valuable for routine use in quality control (QC) laboratories for quantitation of DUV and SEL in their bulk forms and capsules.

Revisiting the HPLC-FLD Method to Quantify Paralytic Shellfish Toxins: C3,4 Quantification and the First Steps towards Validation.

Paralytic shellfish toxins (PSTs) are a large group of biotoxins that cause paralytic shellfish poisoning. Their appearance in natural waters and their ingestion by aquatic species have a huge socio-economic impact, whereby their monitoring is of the upmost relevance to minimize the consequences. For earlier detection and faster response/action by stakeholders, validation of adjusted analytical methods, particularly for lower concentration levels, is important. This work proposes a derived High-Performance Liquid Chromatography method, with fluorescence detection (HPLC-FLD). The main differences from the official method are the size of the HPLC column and the gradient elution conditions. It covers the current eleven certified reference materials (CRM) available on the market, including the most recent—C3,4. This first calibration report for C3,4 suggests limits of detection (LOD) and limits of quantification (LOQ) of 6 nM and 19 nM (~5 µg STX.2HCl eqv./kg and 17 µg STX.2HCl eqv./kg), respectively. For the remaining CRM, LODs ranged between 3 and 28 nM (~0.9 and 127 µg STX.2HCl eqv./kg), while LOQs varied between 11 and 94 nM (~3 and 409 µg STX.2HCl eqv./kg, considering toxicity equivalency factors (TEFs) reported by EFSA).

Application of Solid-Phase Extraction and High-Performance Liquid Chromatography with Fluorescence Detection to Analyze Eleven Bisphenols in Amniotic Fluid Samples Collected during Amniocentesis.

Amniocentesis involves taking a sample of the amniotic fluid in order to perform a karyotype test and diagnose any genetic defects that may affect the fetus. Amniotic fluid has been collected from patients with an indication for amniocentesis in the 15–26th week of pregnancy. A simple and sensitive high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method for identification and quantification of eleven selected bisphenols in amniotic fluid samples is proposed. The proposed method involved protein precipitation using acetonitrile, and next the extraction and concentration of analytes by solid-phase extraction (SPE). The solid-phase extraction (SPE) procedure with application of Oasis HLB SPE columns performed well for the majority of the analytes, with recoveries in the range of 67–121% and relative standard deviations (RSD%) less than 16%. The limits of detection (LODs) and quantification (LOQs) of all the investigated analytes were in the range of 0.8–2.5 ng mL−1 and 2.4–7.5 ng mL−1 (curves constructed in methanol) and 1.1–5.2 ng mL−1 and 3.2–15.6 ng mL−1 (curves constructed in the amniotic fluid), respectively. The method was validated at the following two concentration levels: 10 ng mL−1 (2 × LOQ) and 20 ng mL−1 (4 LOQ). The results confirm the validity of the SPE procedure and HPLC-FLD method for identification and quantification of bisphenols in amniotic fluid samples collected during an amniocentesis. The result obtained show that HPLC-FLD is a useful method for determination of bisphenol residues at nanogram per milliliter concentrations in amniotic fluid samples. Residues of five analytes (BADGE·2H2O, BPAF, BADGE, BADGE·H2O·HCl and BADGE·2HCl) were detected in amniotic fluid samples. Additionally, the harmfulness of bisphenols as potential pathogens that may cause karyotype disorders and contribute to preterm birth was estimated.

Development and Validation of a Rapid and Reliable HPLC-FLD Method for the Quantification of Ciprofloxacin and Enrofloxacin Residues in Zea mays

The accumulation of antibiotics by plants is a currently concern associated to yield reduction and transference of antibiotic residues along the food web. Maize is a staple food in many parts of the world; it is also considered an important ingredient in animal nutrition. Considering the lack of validated analytical methods for the analysis of ciprofloxacin (CIPRO) and enrofloxacin (ENRO) in the shoot of Zea mays maize, we developed a reliable reversed-phase high-performance liquid chromatography method with fluorescence detection (HPLC-FLD). This method was validated for linearity, matrix effects, precision, accuracy, limits of quantification (LOQ) and detection (LOD), and robustness. The analytical curves were linear with coefficient of determination (R2 ) of 0.9907 for CIPRO and 0.9962 for ENRO. The LOD values were 16.65 and 6.57 μg kg−1 for CIPRO and ENRO, respectively, whereas LOQ values were 50.44 μg kg−1 (CIPRO)and 19.92 μg kg−1 (ENRO). HPLC-FLD also displayed good precision and accuracy. Therefore, the proposed method can be considered a reliable and useful tool for the analysis of ciprofloxacin and enrofloxacin in the shoot of maize.

Application of Solid Phase Extraction and High-Performance Liquid Chromatography with Fluorescence Detection to Analyze Bisphenol A Bis (2,3-Dihydroxypropyl) Ether (BADGE 2H2O), Bisphenol F (BPF), and Bisphenol E (BPE) in Human Urine Samples.

In this study, we propose a simple, cost-effective, and sensitive high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) for the simultaneous determination of the three bisphenols (BPs): bisphenol A bis (2,3-dihydroxypropyl) ether (BADGE 2H2O), bisphenol F (BPF), and bisphenol E (BPE) in human urine samples. The dispersive solid phase extraction (d-SPE) coupled with solid phase extraction (SPE) procedure performed well for the analytes with recoveries in the range of 74.3–86.5% and relative standard deviations (RSD%) less than 10%. The limits of quantification (LOQs) for all investigated analytes were in the range of 11.42–22.35 ng mL−1. The method was validated at three concentration levels (1 × LOQ, 1.5 × LOQ, and 3 LOQ). During the bisphenols HPLC-FLD analysis, from 6 min a reinforcement (10 or 12) was used, therefore analytes might be identified in the small volume human urine samples. The results demonstrated clearly that the approach developed provides reliable, simple, and rapid quantification and identification of three bisphenols in a urine matrix and could be used for monitoring these analytes.

Cross-validation of biomonitoring methods for polycyclic aromatic hydrocarbon metabolites in human urine: Results from the formative phase of the Household Air Pollution Intervention Network (HAPIN) trial in India

The Household Air Pollution Intervention Network (HAPIN) trial is evaluating health benefits of a liquefied petroleum gas (LPG) stove intervention in biomass cook-fuel using homes (n = 3200) in four low-and middle-income countries (LMICs) that include Peru, Guatemala, Rwanda and India. Longitudinal urine samples (n = 6000) collected from enrolled pregnant women, infants and older women will be analyzed for biomarkers associated with exposure and health outcomes. We report results from cross-validation of a lower cost high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method with a higher resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the measurement of 1-hydroxypyrene (1PYR) and 2-naphthol (2NAP). Twenty-five split urine samples were analyzed by HPLC-FLD method at the India trial site in Chennai, India and by LC-MSMS method at the trial wide Biomarker Coordinating Center, Emory University, USA. The limits of detection (LOD) for the HPLC-FLD method were 0.02 ng/mL and 0.07 ng/mL for 2NAP and 1PYR, respectively. Bland-Altman analysis estimated a bias of 2.98 ng/ml for 2NAP (95% CI: −5.22, −0.75) and 0.09 ng/mL for 1PYR (95% CI: −0.02, 0.21) with HPLC-FLD levels being lower than LC-MSMS levels at higher concentrations. Analyses of additional urine samples (n = 119) collected during the formative phase of the HAPIN trial in India, showed 2NAP and 1PYR levels to be consistently above the limit of quantification (LOQ) and demonstrated the applicability of the method. The HPLC-FLD method can serve as a cost-effective and reliable analytical method to measure 2NAP and 1PYR in human urine in LMICs, within and beyond the HAPIN trial.

Occurrence, dietary exposure and risk assessment of aflatoxins in arepa, bread and rice

The purpose of this study was to assess the risk of aflatoxins due to arepa, bread and rice consumption among the adult population of the Department of Caldas, Colombia. A high-performance liquid chromatography method with fluorescence detection (HPLC-FD) was validated and used to determine aflatoxins in 246 samples. Dietary exposure was calculated by estimated daily intake (EDI), whereas risk characterization was assessed using the risk of cancer and margin of exposure (MOE) approaches. The method showed good selectivity, linearity (R2 > 0.9993), limit of detection (LOD = 0.004–0.014 ng mL−1), limit of quantification (LOQ = 0.014–0.046 ng mL−1), accuracy (2.40%–14.1%), and recovery (58%–110%). Aflatoxins were detected in 38.6% of the samples at levels between 0.08 and 41.75 μg kg−1, and 11.4% of the samples contaminated with AFB1 exceeded the limit (2 μg kg−1) established by the European Commission (EC). The EDI of aflatoxins due to the consumption of arepa (0.732) and rice (3.093) were above the recommended level of 0.017 ng kg−1 bw day−1. Also, the estimated number of liver cancer cases associated with AFB1 exposure due to arepa (0.726) and rice (3.314) consumption was above the suggested limit of 0.1 cases/75 years/100,000 individuals. Finally, the MOEs obtained by arepa and rice consumption were 277.5 and 60.79, respectively, far from the safe margin of 10,000, thereby indicating a potential risk to public health.

Contamination Parts and Residue Levels of Multi-Mycotoxins in Medicinal and Edible Locust.

Locust is esteemed as a traditional Chinese medicine, as well as one of the most important nutritional foods especially in Asian countries. However, some toxic secondary metabolites such as mycotoxins are usually found in different parts of locust to affect its quality and safety. This study aimed to investigate the aflatoxins (AFs) contaminated parts by observing Aspergillus flavus, spores’ diameter, amount and distribution on head, tentacle, wing, belly and shank parts of the locust with scanning electron microscopy (SEM). Furthermore, to assess the residue levels of multi-mycotoxins in the locust, the high performance liquid chromatography with fluorescence detection (HPLC-FLD) was adopted. The technique was used to determine the contents of AFs, zearalenone (ZON) and α-zearalenol (α-ZOL) in locust and the positive samples were confirmed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The chromatographic conditions, MS/MS parameters and the method of sample extraction were carefully optimized. Results revealed that obvious differences of Aspergillus flavus strains and spores were found, while the spores’ diameter ranged from 3.0 to 13.0 μm in different contaminated parts of the locust samples. The HPLC-FLD method for multi-mycotoxins analysis showed good selectivity, linearity, recovery and precision. Limits of quantification (LOQs) were lower than 27.6 μg/kg, while limits of detection (LODs) were in the range of 0.02–8.6 μg/kg. The accuracy of the developed method was validated regarding recoveries of 80.1–118.1% with relative standard deviation (RSD) ≤ 11.4%. Finally, the developed multi-mycotoxin method was applied for screening of these mycotoxins in 11 commercial locust samples. Only AFB1 and AFB2 were found in six samples, and the contamination levels ranged from 0.12 to 4.4 μg/kg, which were lower than the maximum residue limit and can be used safely. This is the first report on the exploration of contamination parts and levels of multi-mycotoxins in medicinal and edible locust. The combined method of SEM and HPLC-FLD exhibited advantages of low cost, high sensitivity, rapid determination, convenience and especially intuitive judgment, which is proposed for contamination parts observation, for the large-scale quantification of multi-mycotoxins in other medicinal animal matrices.

An Improved HPLC-FLD for Fast and Simple Detection of Ethyl Carbamate in Soy Sauce and Prediction of Precursors

A high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method for ethyl carbamate (EC) determination in Chinese soy sauce was developed and used to predict EC precursors, and extraction, derivatization, and chromatographic conditions were optimized systematically. Under optimal conditions, the limit of detection, linear range, and recoveries were 3.91 μg/L, 12.87–274.87 μg/L, and 81.5–95.4%, respectively. The method precision was less than 8.9% (RSD) and no significant difference was found between EC determinations measured by HPLC-FLD and GC/MS. Using the proposed HPLC-FLD method, the EC contents in Chinese soy sauces ranged from not detected to 64.88 μg/L (n = 19). Except for one soy sauce brand, the EC contents in low-salt solid-state fermented (LSF) soy sauces were lower than those in high-salt liquid fermented (HLF) soy sauces and were mainly affected by alcohol content. Moreover, the free ornithine and total arginine contents were significantly correlated with EC content in the soy sauce products. The results of this work provide a foundation for further study of EC formation and inhibition in Chinese soy sauces.

Contamination of smoked meat and fish products from Polish market with polycyclic aromatic hydrocarbons

The aim of this work was to determine the level of contamination of different kinds of commercial smoked meat and fish products from Polish market with polycyclic aromatic hydrocarbons (PAHs) for which the limiting values are established in the Commission Regulation (EU) 2015/1125. These PAHs are benzo(a)pyrene (BaP) and the sum of the following four PAHs: benzo(a)pyrene, benzo(a)anthracene (BaA), benzo(b)fluoranthene (BbFA) and chrysene (Chr). The research materials were fish, meat and chicken products smoked by the industrial or traditional techniques. The research methods included process of saponification of the samples, extraction of the polycyclic aromatic hydrocarbons fraction, and then qualitative and quantitative determination of PAHs by high performance liquid chromatography with fluorescence detection method (HPLC-FLD). The highest BaP content and the sum of the four polycyclic aromatic hydrocarbons (4PAHs) (36.51 mg/kg and 73.01 mg/kg, respectively) were found in canned smoked sprats in oil and these values exceeded the maximum levels given in the Commission Regulation (EU) 2015/1125. The analyzed products smoked by traditional technique contained significantly more PAHs as compared to the products smoked by the industrial technique or produced with smoke flavourings.

Analysis of serotonin concentrations in human milk by high-performance liquid chromatography with fluorescence detection

Serotonin (5-hydroxytryptamine, 5-HT) plays an important role in milk volume homeostasis in the mammary gland during lactation; 5-HT in milk may also affect infant development. However, there are few reports on 5-HT concentrations in human breast milk. To address this issue, we developed a simple method based on high-performance liquid chromatography with fluorescence detection (HPLC-FD) for measuring 5-HT concentrations in human breast milk. Breast milk samples were provided by four healthy Japanese women. Calibration curves for 5-HT in each sample were prepared with the standard addition method between 5 and 1000 ng/ml, and all had correlation coefficients >0.999. The recovery of 5-HT was 96.1%–101.0%, with a coefficient of variation of 3.39%–8.62%. The range of 5-HT concentrations estimated from the calibration curves was 11.1–51.1 ng/ml. Thus, the HPLC-FD method described here can effectively extract 5-HT from human breast milk with high reproducibility.

The pharmacokinetics, bioavailability and excretion of bergapten after oral and intravenous administration in rats using high performance liquid chromatography with fluorescence detection.

A sensitive, specific, reproducible and optimized high performance liquid chromatography with fluorescence detection (HPLC-FLD) method for the determination of bergapten in rat plasma was established and applied to the pharmacokinetic and bioavailability study in rat after oral and intravenous administration of bergapten. The method was also successfully applied to the excretion study of bergapten after an oral administration of bergapten at a dose of 15 mg kg−1 to rats. The sample preparation was achieved using liquid–liquid extraction. Isoimperatorin was used as the internal standard (IS). The analytes were detected by using fluorescence detection at an excitation and emission wavelength of 288 and 478 nm, respectively. Using aqueous formic acid (0.1 %, v/v) and acetonitrile as the mobile phase, the chromatographic separation was achieved on a Hedera™ ODS column at a flow rate of 1 mL min−1. The lower limit of quantitation (LLOQ) of bergapten was 2 ng mL−1. The HPLC-FLD method was successfully applied to the pharmacokinetic, bioavailability and excretion study of bergapten in rats.Graphical abstractAn high performance liquid chromatography with fluorescence detection (HPLC-FLD) method for the pharmacokinetic and bioavailability study in rat after administration of bergapten.

Systematic optimization of an SPE with HPLC-FLD method for fluoroquinolone detection in wastewater

This paper describes a selective and ultra-sensitive analytical method for simultaneous determination of 11 fluoroquinolone (FQ) antibiotics in environmental and wastewater samples. The method employs offline solid-phase extraction (SPE) and reversed-phase high performance liquid chromatography with fluorescence detection (HPLC-FLD). A weak cation exchange SPE protocol was developed with a novel loading volume optimization algorithm and a methanol cleanup step to remove background organic matter. Various parameters were optimized to recover FQs from water/wastewater and analyte recovery was generally greater than 80%. Chromatographic separation of the 11 FQs was achieved on a 150mm pentafluorophenyl column using a gradient elution scheme with methanol, acetonitrile, and 20mM phosphate buffer (pH=2.4). Excitation and emission wavelengths were individually optimized for each FQ using fluorescence spectroscopy; the excitation and emission wavelengths were 276–296nm and 444–506nm, respectively. Instrumental quantitation limits were 20–100pg of mass injected. Of the 11 FQs investigated, seven (i.e., ciprofloxacin, difloxacin, enrofloxacin, fleroxacin, norfloxacin, moxifloxacin, and ofloxacin) were detected during a four-month sampling campaign of wastewater and wastewater-impacted surface water. Concentrations of FQs in raw wastewater, wastewater effluent, and wastewater-impacted surface water were 5–1292, 2–504, and 4–187ng/L, respectively.

Multi-mycotoxin determination in baby foods andin vitro combined cytotoxic effects of aflatoxin M1 and ochratoxin A

The co-occurrence of mycotoxins in baby foods, including aflatoxin M1(AFM1) and ochratoxin A (OTA), has been reported in previous studies, but data on their potential combined toxic effects are still missing. The present work aimed at (1) validating an in-house multi-mycotoxin high performance liquid chromatography with fluorescence detection (HPLC-FLD) method for AFM1, total aflatoxins (aflatoxin B1(AFB1), aflatoxin B2(AFB2), aflatoxin G1(AFG1), aflatoxin G2(AFG2)) and OTA in infant formulae (milk powders) and cereal baby foods (flours), and (2) assessing the combined cytotoxic effects of AFM1and OTA in an intestine-derived cell line. The HPLC-FLD method, which included a chloroform extraction, liquid-liquid extraction, immunoaffinity column clean-up and fluorescence detection after post-column derivatisation with electrochemically generated bromine, was adequate for the analysis of baby foods and met the requirements of validation and quality control for the studied working ranges. The limits of quantification for AFM1, AFB1, AFB2, AFG1, AFG2and OTA were 0.069, 0.032, 0.020, 0.047, 0.020 and 0.244 μg/kg, respectively. The mean recovery values were 96, 114, 112, 107, 101 and 87%, respectively. A dose-dependent cytotoxicity was observed for individual and combined AFM1and OTA using the Caco-2 cell line, which represents a site of contact of both mycotoxins in the body, after oral exposure. Interactions between both mycotoxins were disclosed by application of the concentration addition (CA) and independent action (IA) models, revealing the predominance of an antagonistic pattern. In conclusion, this study proposes a HPLC-FLD method for multi-mycotoxin monitoring in baby foods and provides evidence for the interaction between AFM1and OTA, and for the applicability of CA/IA models to predict the effect of mycotoxins mixtures, further contributing to the prevention of mycotoxins-associated adverse health effects.

HPLC method with fluorescence detection for the determination of ligustilide in rat plasma and its pharmacokinetics

Context: Few methods have been reported for the quantification of ligustilide (LIG) in biosamples: the pretreatment of the biological samples were laborious and time-consuming.Objective: A high-performance liquid chromatographic method with fluorescence detection (HPLC-FLD) for the determination of LIG in rat plasma was developed and validated. Pharmacokinetics and bioavailability of LIG were determined by systematic investigation in Sprague-Dawley rats.Materials and methods: LIG was isolated from the volatile oil of Radix Angelica sinensis and further purified by silica gel column chromatography. Podophyllotoxin was used as an internal standard. The analytes were detected by using fluorescence detection at an excitation and emission wavelength of 290 and 395 nm during 0–4 min, and 336 and 453 nm during 4–14 min, respectively. LIG pharmacokinetics was studied in rats after oral and intravenous administration of 12.5, 25 and 50 mg/kg doses.Results: Two calibration curves (Y = 133.49 X − 14.27 (r = 0.9995), Y = 145.61 X + 13.76 (r = 0.9996)) were constructed in the range of 2.44–10 000 ng/mL for LIG with a lower limit of quantitation of 2.44 ng/mL. Both intra-day and inter-day precision were less than 6%. Accuracy ranged from 88.93 to 99.52%. The recovery ranged from 89.07 to 99.71%. The absolute bioavailability values were 71.36, 68.26 and 75.44% for oral doses of 12.5, 25 and 50 mg/kg, respectively.Conclusion: The present HPLC-FLD method was rapid, sensitive and reliable. LIG was absorbed and eliminated rapidly in rat.

Determination of macrocyclic lactones in food and feed

A confirmatory high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) was developed and validated for the simultaneous determination of the following anti-parasitic veterinary drugs in foodstuffs (liver, muscle and milk) and feed: abamectin, doramectin, emamectin, eprinomectin, ivermectin and moxidectin. Samples were extracted, purified and analysed by HPLC-FLD after prior derivatisation of analytes with N-methylimidazole, trifluoracetic anhydride and acetic acid to produce stable fluorescent derivatives. Recoveries were in the range of 73–98% (liver), 82–93% (muscle), 77–84% (milk), and 93–110% (feed). In all matrices the coefficients of variation for repeatability and intra-laboratory reproducibility were satisfactory. For emamectin in liver the worst method performance was observed. As this multi-analyte method fully met the required criteria and applicability both to food and feed, it has been successfully applied in National Control Plans in food and feed.

Application of quality by design concept for the optimization of an analytical method for rhubarb anthraquinones in rat plasma

A robust high performance liquid chromatography with fluorescence detection method (HPLC-FLD) was developed for the determination of five rhubarb anthraquinones in rat plasma using a quality by design approach. A Plackett-Burman design was utilized to screen the effects of methanol content, the pH value of mobile phase, the flow rate, column temperature, and injection volume on peak resolution, number of the theoretical plate, retention time of the last eluted peak, and tailing factor. The results showed that the methanol content in mobile phase, flow rate, and column temperature were statistically significant (p < 0.05). A Box-Behnken experimental design with response surface methodology (RSM) was then utilized to evaluate the effects of these three factors on the selected responses. Derringer' s desirability function was used for the evaluation of the chromatographic goals. The optimum conditions for separation were as follows: an isocratic mobile phase consisting of methanol/0.1% H3PO4 (81.4/18. 6, v/v), a flow rate of 1.1 mL/min, and a column temperature of 31 degrees C. The excitation and emission wavelengths were 440 nm and 540 nm, respectively. The proposed method showed good prediction ability. The results clearly showed that quality by design concept could be effectively applied to optimize the HPLC method.

Determination of serum aromatic amino acid in patients with CRI by high performance liquid chromatography with fluorescence detection

Objective A HPLC-FLD method was developed to determine the levels of serum AAA in CRI patients, and to study the variation of serum AAA in CRI patients and its clinical significances. MethodsSerum samples were collected from 100 health controls and 80 CRI patients. According to 2002 National Kidney Foundation (NKF) staging diagnosis, CRI patients included 4 of stage 2, 12 of stage 3, 12 of stage 4, 52 of stage 5. According to pathogenesis, CRI patients were also divided into 3 groups :chronic nephritis group ( n = 32), DM group ( n = 36), hypertension group ( n = 12 ).Serums were deproteinized by equal volume of 5％ (v/v) PCA and supernate were analyzed direcdy. External standard method was used as quantitative method. The analytical column was Megres C18. 10％ acetonitrile in water was used as mobile phase. Flow rote was 1.0 ml/min. The wavelengths of fluorescence excitation and emission were changed with specific time. The levels of Tyr, Phe and Trp in CRI groups, different CKD stages and different pathogenesis were compared with healthy control groups to evaluated the sensitivity and specificity of serum AAA for CRI diagnosis. Results The linear ranges of the method were 0. 550 -275.000, 3. 050 - 1220. 000 and 0. 049 -49. 000 pμmol/L for Tyr, Phe and Trp, respectively. The limit of detection (LOD) was 0.014 μmol/L for Tyr, 0.500μmol/L for Phe, and 0.005 μmol/L for Trp. The average recovery was 100. 9％, 101.3％ and 98. 5％ for Tyr, Phe and Trp, respectively. Intra-day CVwas 3. 18％ -4. 20％ ( mean was 3. 13％ )and inter-day CV was 3. 18％ -4. 20％ ( mean was 3. 58％ ). The concentration of serum AAA, Tyr and Trp and the ratio of Tyr/Phe in CRI patients were( 135.74 ±23.23 )μmol/L, (52.27 +8.25) μmol/L, (21.49 ±4.25) μmol/L and[0.87(0.68 - 1.05)]μmol/L. which were lower than that in healthy groups (t value was -14. 709, 4.452, 22. 100, U value was 266.000,respectively, P＜0. 05). The concentration of serum AAA, Tyr and Trp and the ratio of Tyr/Phe in healthy groups were ( 174. 47 ± 11.57 ) μmol/L, ( 63.53 ± 4. 68 ) μmol/L, (44. 22 ± 3. 67 ) μmol/L and[0. 97(0. 94 - 1.00)]μmoL/L. There were no statistically significant difference between the different stage of CRI. Compared with the concentration of Tyr, Phe and Trp among chronic nephritis group, DM group,hypertension group, the concentration of Tyr had no significant changes among these three kinds of diseases (P ＞ 0. 05 ). The concentration of Phe had significant changes between Chronic nephritis group and DM group, Chronic nephritis group and hypertension group ( U = 395.00, 114. 00, P ＜ 0. 05 ) ; the concentration of Trp haad significant changes between Chronic nephritis group and DM group ( U = 349.00, P ＜ 0. 05 ).The diagnostic sensitivity and specificity of serum AAA for CRI were 90％ (72/80) and 100. 0％ (100/100).Conclusions The method of high-performance liquid chromatography with fluorescence detection ( HPLC-FLD) is simple, rapid, sensitive and specific. Simultaneous determination of serum AAA was benefit to the diagnosis and evaluation of CRI patients. Key words: Renal insufficiency, chronic; Amino acids, aromatic; Chromatography, high pressure liquid

Quantification of aesculin in rabbit plasma and ocular tissues by high performance liquid chromatography using fluorescent detection: Application to a pharmacokinetic study

A simple and sensitive high performance liquid chromatography method with fluorescence detection (HPLC-FD) was described for the determination of aesculin (AL) at low concentrations in rabbit plasma and ocular tissues. After deproteinization by methanol using pazufloxacin mesilate (PM) as an internal standard (I.S.), supernatants were evaporated to dryness at 40 °C under a gentle stream of nitrogen. The residue was reconstituted in mobile phase and a volume of 20 μL was injected into the HPLC for analysis. Analytes were separated on an Ultimate XB-C18 column (250 mm × 4.6 mm i.d., 5 μm particle size) and protected by a ODS guard column (10 mm × 4.0 mm i.d., 5 μm particle size), using acetonitrile–0.1% triethylamine in water (adjusted to pH 3.0 using phosphoric acid) (12:88, v/v) as mobile phase with a flow rate of 1.0 mL/min. The wavelengths of fluorescence detector (FD) were set at 344 nm for excitation and 466 nm for emission. The lower limit of quantitation (LOQ) for AL was 0.80 ng/mL for plasma and vitreous body, 1.59 ng/mL for aqueous humor, and 6.55 ng/g for iris and 1.66 ng/g for retina. The method was used in the study of AL concentrations in plasma and ocular tissues after topical administration of AL eye drops.

Rapid resolution liquid chromatography–mass spectrometry and high-performance liquid chromatography-fluorescence detection for metabolism and pharmacokinetic studies of ergosta-4,6,8(14),22-tetraen-3-one

Ergosta-4,6,8(14),22-tetraen-3-one (ergone) from many medicinal plants has been demonstrated to possess a variety of pharmacological activities in vivo and in vitro, including cytotoxic, diuretic and immunosuppressive activity. Metabolism and pharmacokinetic studies on rat were conducted for ergone. Rapid resolution liquid chromatography with atmospheric pressure chemical ionization tandem multi-stage mass spectrometry (RRLC-APCI-MS n) and high-performance liquid chromatography with fluorescence detection (HPLC-FLD) methods were applied for the identification and quantification of ergone and its metabolite from rat plasma, faeces and urine. A metabolite was identified by RRLC-DAD-APCI-MS n: 22,23-epoxy-ergosta-4,6,8(14)-triaen-3-one (epoxyergone). The concentrations of the analyte with its metabolites were determined by HPLC-FLD at excitation wavelength of 370 nm and emission wavelength of 485 nm. The samples were deproteinized with methanol after addition of camptothecin as internal standard (IS). The analysis was performed on a Diamonsil C18 column (150 mm × 4.6 mm × 5 μm) with a mobile phase gradient consisting of methanol and water at a flow rate of 1 mL min −1. The assay was linear over the concentration range of 42–1500, 36–7500 and 42–1500 ng mL −1 for plasma, faecal homogenate and urine respectively. The absolute recoveries were found to be 97.0 ± 1.2%, 98.1 ± 0.7% and 96.6 ± 1.8% for plasma, faecal homogenate and urine respectively. The intra-day and inter-day relative standard deviations (RSD) were less than 10%. The previous HPLC–MS/MS method is not affordable for most laboratories because of the specialty requirement and high equipment cost. However, the HPLC-FLD method is economic and operating simply for quantitative determination of ergone and its metabolite in rat plasma, faeces and urine. In addition, liquid chromatography coupled with ion trap multi-stage mass spectrometry is becoming a useful technique for ergone metabolite identification.