Ion-pair RP-HPLC Research Articles

Environmentally Sustainable Achiral and Chiral Chromatographic Analysis of Amino Acids in Food Supplements.

Two LC methods were developed for the achiral and chiral reversed-phase (RP) analysis of an amino acid (AA) pool in a food supplement, in compliance with the main paradigms of Green Chromatography. A direct achiral ion-pairing RP-HPLC method was optimized under gradient conditions with a water-ethanol (EtOH) eluent containing heptafluorobutyric acid (0.1%, v/v), to quantify the eight essential AAs (Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val) contained in the food supplement. Thus, the usually employed acetonitrile was profitably substituted with the less toxic and more benign EtOH. The method was validated for Leu and Phe. The chiral LC method performed with a teicoplanin chiral stationary phase was developed with a water-EtOH (60:40, v/v) eluent with 0.1%, v/v acetic acid. The enantioselective analysis was carried out without any prior derivatization step. Both developed methods performed highly for all eight AAs and revealed that: (i) the content of six out of eight AAs was consistent with the manufacturer declaration; (ii) only L-AAs were present. Furthermore, it was demonstrated that a two-dimensional achiral-chiral configuration is possible in practice, making it even more environmentally sustainable. A molecular modelling investigation revealed interesting insights into the enantiorecognition mechanism of Lys.

Determination of individual oligonucleotide impurities by small amine ion pair-RP HPLC MS and MS/MS: n − 1 impurities

Ion pair-reversed phase (IP-RP) HPLC is one of the most widely used methods for the analysis of oligonucleotide impurities. The method is compatible with mass spectrometry and has been used to guide the development of improved synthesis and purification approaches. The ability to detect and characterize impurities depends on the reagents and the IP buffer system employed, as each can directly affect the degree of chromatographic separation and the sensitivity of detection by MS. Previous work in our laboratory has shown that small alkyl amines are suitable IP reagents for the analysis of impurities in phosphate diester oligonucleotides and can be used to differentiate among individual members of composite impurity families. The addition of an alkyl acid often further enhances peak separation, but at the detriment of ion signal. An improved method with increased chromatographic performance and sensitivity of detection is presented here. Improvements were mainly realized through the use of lower concentrations of small alkyl amine (i.e., 5 mM) and acid (0.5 mM) IP reagents, and ammonium bicarbonate (20 mM) as a buffer. The improved capabilities of the new method are demonstrated by separation of the individual components of the composite n − 1 impurity in a set of four production-scale batches of a single oligonucleotide. Addition of the alkyl acid resulted in resolution of most individual n − 1 impurities. The observed enhanced sensitivity of detection allowed multiple reaction monitoring (MRM) experiments, which were used to differentiate among unresolved impurities.

Development and Validation of a Stability-Indicating Ion-Pair RP-HPLC Method for Determination of Bethanechol with UV Detection: Application to Pharmaceutical Analysis

A rapid and stability indicating ion-pair RP-HPLC method was developed for estimation of bethanechol chloride (BC) in pharmaceutical preparations. The separation was achieved on a phenyl column using a mobile phase consisting of 0.05 M phosphate buffer (pH 6) – ethanol (98:2 v/v) mixture containing 0.56 mg/mL of sodium 1-heptanesulfonate. Quantitation was achieved with UV detection at 190 nm. In forced degradation studies, the drug was subjected to oxidation, hydrolysis, photolysis, and heat. The method was validated for specificity, selectivity, linearity, precision, accuracy, and robustness. The analytical procedure was found to be linear in a BC concentration range of 5.0 – 500.0 μg/mL (r2 = 0.9997). Precision was evaluated by replicate analysis in which%deviations from relative standard values of peak areas were found below 2.0. The recoveries obtained (99.0 – 102.0%) ensure the accuracy of the proposed method. Impurities as well as excipients were well resolved from the parent drug. Accordingly, the proposed validated procedure was proved to be suitable for routine and stability analysis of BC in pharmaceutical preparations.

Experimental design methodology for optimization and robustness determination in ion pair RP-HPLC method development: Application for the simultaneous determination of metformin hydrochloride, alogliptin benzoate and repaglinide in tablets

Experimental design methodology (DOE) surpasses traditional (one variable at a time) approach in improving the chromatographic separation performance with minimum effort and resources. Since combination therapy of oral hypoglycemic drugs was generally recommended by Clinical Practice Guidelines on hypoglycemic agent therapy for treatment of type 2 diabetes mellitus, DOE was adopted in development of an ion pair RP-HPLC method for the simultaneous determination of metformin hydrochloride (MET), alogliptin benzoate (ALO) and repaglinide (REP) in combined binary tablets. Screening using Plackett-Burman design followed by face centered composite design enabled the estimation of optimum settings that accomplish the most appropriate resolution and adequate peak shape within suitable run time. The designed models were statistically analyzed, graphically presented by surface plots and the relationships between coefficients of the derived polynomial equations were interpreted. The models fitted data adequately and could be used for response prediction. Chromatographic separation was achieved using Inertsil ODS column (250 mm, 4.6 mm, 5 μm), acetonitrile: phosphate buffer (0.01 M, adjusted to pH 2.5 with o-phosphoric acid): 0.3% sodium heptane sulfonate in water (60:20:20, v/v/v) as a mobile phase at flow rate 1 mL min−1. UV detection was carried out at 220 nm. Sharp and well separated peaks for the cited drugs were obtained. Laboratory prepared mixtures containing MET, ALO and REP were analyzed with mean percentage recoveries 99.66 ± 0.468, 99.98 ± 0.398 and 99.70 ± 0.988, respectively. The method was successfully applied for the determination of the drugs in binary tablets with good recoveries of 99.75 ± 0.6 and 99.74 ± 0.982 for MET and ALO tablets and 99.76 ± 0.619 and 100.08 ± 1.159 for MET and REP tablets. Method validation was performed according to ICH guidelines Q2 (R1). Robustness was evaluated using the created models. The greenness profile of the developed method was evaluated using the analytical Eco-scale and Green Analytical Procedure Index as assessment tools. The developed method proved to be accurate, selective, precise and ecofriendly procedure for the determination of the cited drugs in binary tablets and could be applied in quality control laboratories for routine analysis of the drugs.

Stress Testing of Quetiapine Fumarate by a Chemometric Assisted Multi-Component Ion-Interaction System

Classical experimental designs have been popularly employed in establishing robust analytical methods while achieving other advantages, viz., reduction in the number of experiments and hence lower reagent consumption and less laboratory work. To achieve optimum chromatographic condition, a computer-aided Box-Behnken Design (BBD) in ion-pairing stability-indicating RP-HPLC assay of quetiapinefumarate along with its stress related substances has been investigated here, proving to be an invaluable tool in ascertaining a reliable method. The study includes forced degradation of quetiapinefumarateunder acidic, alkaline, photo, oxidative and peroxide stress conditions followed by separation of degradation products. Critical factors including buffer pH, % organic phase (acetonitrile) and concentration of hexane sulphonate (ion-pairing reagent) susceptible to influence the separation (critical resolutions) and total analysis time were investigated by response surface methodology. The best optimal separation condition as obtained was observed on an enable C-18 column (250mm x 4.6mm i.d, 5μm particle size) using mobile phase composed of Phosphate buffer (pH 2.0) containing 0.002mM hexane sulphonate and acetonitrile (74.4:25.6 v/v) at a flow rate of 1.00ml/min. The eluents were observed at 220nm using a PDA detector. Further, the method was validated to ensure its reliability and other regulatory criteria are met.

Development, Validation and Application of RP-HPLC Method: Simultaneous Determination of Antihistamine and Preservatives with Paracetamol in Liquid Formulations and Human Serum.

In this article we describe development and validation of stability indicating, accurate, specific, precise and simple Ion-pairing RP-HPLC method for simultaneous determination of paracetamol and cetirizine HCl along with preservatives i.e. propylparaben, and methylparaben in pharmaceutical dosage forms of oral solution and in serum. Acetonitrile: Buffer: Sulfuric Acid (45:55:0.3 v/v/v) was the mobile phase at flow rate 1.0 mL min-1 using a Hibar® Lichrosorb® C18 column and monitored at wavelength of 230nm. The averages of absolute and relative recoveries were found to be 99.3%, 99.5%, 99.8% and 98.7% with correlation coefficient of 0.9977, 0.9998, 0.9984, and 0.9997 for cetirizine HCl, paracetamol, methylparaben and Propylparaben respectively. The limit of quantification and limit of detection were in range of 0.3 to 2.7 ng mL-1 and 0.1 to 0.8 ng mL-1 respectively. Under stress conditions of acidic, basic, oxidative, and thermal degradation, maximum degradation was observed in basic and oxidative stress where a significant impact was observed while all drugs were found almost stable in the other conditions. The developed method was validated in accordance with ICH and AOAC guidelines. The proposed method was successfully applied to quantify amount of paracetamol, cetirizine HCl and two most common microbial preservatives in bulk, dosage form and physiological fluid.

Based on Nucleotides Analysis of Tumor Cell Lines to Construct and Validate a Prediction Model of Mechanisms of Chemotherapeutics.

Cancer is one of the diseases that seriously threaten to human life worldwide. Up to now, chemotherapy remains to be a critical means of cancer treatment, thus the development of chemotherapeutical drugs has become a top priority. An ion pair high performance liquid chromatography (ion pair RP-HPLC) was established for analyzing intracellular nucleotides of tumor cell lines. In this article, a partial least-squares discriminant analysis (PLS-DA) prediction model of mechanisms of chemotherapeutics was established based on four types of drugs with different mechanisms, including antimetabolic agents, antineoplastic agents that affect protein synthesis, agents directly acting on DNA, and RNA interference agents. Then four anti-tumor agents commonly used in clinical were used to validate the availability of the prediction model. Three natural compounds, including 16- dehydropregnenolone (16-DHP), apigenin (API) and diosgenin (DIO), were reported to display anti-tumor effect with unclear mechanisms. The three components were applied to this prediction model firstly. In conclusion, the recognition model was proved to be accurate and feasible to some degree and might become a promising auxiliary method in the process of chemotherapeutic drugs development.

Effects of antibiotic antitumor drugs on nucleotide levels in cultured tumor cells: an exploratory method to distinguish the mechanisms of antitumor drug action based on targeted metabolomics

Nucleotide pools in mammalian cells change due to the influence of antitumor drugs, which may help in evaluating the drug effect and understanding the mechanism of drug action. In this study, an ion-pair RP-HPLC method was used for a simple, sensitive and simultaneous determination of the levels of 12 nucleotides in mammalian cells treated with antibiotic antitumor drugs (daunorubicin, epirubicin and dactinomycin D). Through the use of this targeted metabolomics approach to find potential biomarkers, UTP and ATP were verified to be the most appropriate biomarkers. Moreover, a holistic statistical approach was put forward to develop a model which could distinguish 4 categories of drugs with different mechanisms of action. This model can be further validated by evaluating drugs with different mechanisms of action. This targeted metabolomics study may provide a novel approach to predict the mechanism of action of antitumor drugs.

Simultaneous determination of ascorbic acid, aminothiols, and methionine in biological matrices using ion-pairing RP-HPLC coupled with electrochemical detector.

A novel highly sensitive ion-pairing reversed-phase high performance liquid-chromatography/electrochemical detection method for simultaneous determination of L -ascorbic acid, aminothiols, and methionine in biological matrices is presented. Reduced forms of the analytes are extracted from sample matrices with 10% m-phosphoric acid solution(aqueous). To determine the total vitamin C, the total aminothiols, and the total methionine, samples are treated with tris(2-carboxyethyl)phosphine solution in 0.05% trifluoroacetic acid solution(aqueous) subsequent to deproteination to reduce the oxidized forms of these compounds. Various analytes are separated on a C18 (250 × 4.6 mm, 5 μm) analytical column using methanol-0.05% trifluoroacetic acid solution(aqueous) (05:95 v/v, containing 0.1 mM 1-octane sulfonic acid as the ion-pairing agent) as the isocratic mobile phase that is pumped at a flow rate of 1.5 ml/min at room temperature. The column eluents are monitored at a voltage of 0.85 V. These analytes are efficiently resolved in less than 20 min using n-acetyl cysteine as the internal standard.

Ion-pair RP-HPLC method for the estimation of eberconazole nitrate in bulk form and in pharmaceutical dosage forms

Summary An isocratic ion-pair reversed phase high-performance liquid chromatography-ultraviolet (RP-HPLC-UV) method for analysis of eberconazole nitrate in bulk and in pharmaceutical dosage forms has been developed and validated. Best separation was achieved on Lichrospher C18 column (250 mm × 4.6 mm, 5 μm) using a mobile phase of 10 mM potassium dihydrogen phosphate containing 10 mM tetra-butyl ammonium hydroxide (pH adjusted to 2.8 with ortho phosphoric acid) and methanol (75:25, v/v) at a flow rate of 1.0 mL min−1. UV detection was performed at 220 nm. The method was validated for specificity, linearity, precision, accuracy, limit of detection, limit of quantification, robustness, and solution stability. The calibration plot was linear over the concentration range of 10–80 μg mL−1 (r2 = 0.999) and the limits of detection and quantification were 0.3 and 0.9 μg mL−1, respectively. Intra-day and inter-day precisions were 1.13% and 1.67%, respectively. Experimental design was employed to optimize the metho...

Combined monodimensional chromatographic approaches to monitor the presence of d-amino acids in cheese

The presence of d-amino acids (d-AAs) as a consequence of natural or artificial interventions such as ageing, microorganism action, preservative and conservative processes (alkali or heat treatment), is a scarcely treated aspect from the scientific community. It is also fully documented that even a minor degree of racemisation on the proteins' AAs is the cause of a reduced digestion of such proteins. Besides interfering with the regular metabolism of l-AAs, d-AAs can also contribute to the development of pathological conditions in humans. So far, nearly all the most important chromatographic techniques were applied to quantify d-AAs in foodstuffs. However, most of them rely upon pre- or post-column derivatization procedures, often combined with sophisticated analytical equipments. Differently, in this paper we propose an easy-to-set up combination of monodimensional chromatographic methods to monitor the variation of the d-Ala, d-Asp and d-Glu content in two commercially available Spanish cheese samples prepared from the same milk mixture and characterized by a different maturity time: no ripening and six months ripening. After the free amino acid mixture was extracted from the two cheese samples, an ion-pairing RP-HPLC achiral protocol was firstly optimized with the objective to avail of a method enabling the complete distinction of Ala, Asp, and Glu from all the other aminoacidic species in the two extracts. An ion-exchange-based chromatographic method was also optimized, thus allowing a profitable fractionation of the two aminoacidic mixtures. With such a procedure, less complex samples to be analyzed with a chiral ligand-exchange chromatography (CLEC) stationary phase based on S-trityl-l-cysteine (l-STC) units were obtained.The optimized CLEC conditions were then applied to the previously identified Ala, Asp and Glu containing fractions as well as to those including all the remaining species. For all the three compounds the enantiomeric excess (ee) was found to decrease passing from the ripened to the fresh cheese. As expected, the largest difference was found for Ala (ee value from 83.0% down to 20.5%), followed progressively by Asp (ee value from 90.5 to 75.0%) and Glu (ee value from 99.0 to 91.8%).

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY, TLC-DENSITOMETRY, AND FIRST-DERIVATIVE SPECTROPHOTOMETRY FOR SIMULTANEOUS DETERMINATION OF AMLODIPINE AND PERINDOPRIL IN BULK POWDER AND ITS TABLETS

Three simple, sensitive, and specific methods were developed for simultaneous determination of amlodipine besylate (AML) and Perindopril Erbumine (PER) without previous separation. The first method was dependent on the first derivative of the ratio spectra by measuring the amplitudes at 348 nm for amlodipine using 50 µg mL−1 of perindopril as a divisor and at 227 nm for perindopril using 30 µg mL−1 of amlodipine as a divisor. The second method was based on ion-pair RP-HPLC. Satisfactory resolution was achieved using RP-C18 chromatographic column Zorbax Extend column and a mobile phase consists of potassium dihydrogen phosphate buffer (0.05 M, pH 3.0 ± 0.02 adjusted by orthophosphoric acid): acetonitrile 30:70 v/v at a flow rate 1 mL/min using 0.002 M sodium heptanesulfonate in the aqueous phase. UV detection was performed at 215 nm. The third method was based on TLC; the separation was carried out on Fluka TLC aluminum sheets silica gel 60 F254, using n-butanol:water:glacial acetic acid (4:5:1, v/v/v) as the mobile phase. The validation of the proposed methods was applied according to ICH guidelines and LOD and LOQ were calculated. The suggested methods were successfully applied for the determination of the cited drugs in bulk powder and commercial tablets.

EFFECTIVE SEPARATION AND SIMULTANEOUS DETECTION OF LEVOFLOXACIN, CEFAZOLIN, AND CEFMINOX IN HUMAN URINE BY RP-HPLC

The combination of some drugs can enhance pharmacodynamics. Levofloxacin (LOFX), cefazolin (CFZL), and cefminox (CMNX) can be combined and administered perorally or intravenously and are excreted mainly into the urine. A new ion-pair RP-HPLC method was developed, for the first time, for effective separation and simultaneous determination of the three drugs in human urine. The analytical conditions were investigated and optimized. Under optimal conditions, the three drugs can be separated effectively. Good linearity was achieved in 0.24–50 µg/mL for LOFX, 15–1500 µg/mL for CFZL, and 0.6–60 µg/mL for CMNX, with the correlation coefficients(r) of >0.999. The LOD values for LOFX, CFZL, and CMNX were 0.08, 3.75, and 0.10 µg/mL, respectively. The recoveries ranged from 110 to 115% with the RSD of 2.4–3.9% for LOFX, from 101 to 115% with the RSD of 2.2–3.2% for CFZL, and from 94.7 to 103% with the RSD of 1.0–3.2% for CMNX. The proposed method is effective, accurate, and provides the sensitivity and linearity necessary for analysis of the studied drugs in human urine at clinically relevant concentrations.

Elution behavior of short dsDNA strands in silicon micropillar array columns in ion pair reversed‐phase chromatography mode

In the present paper, dsDNA separation has been studied in a silicon micropillar array column using ion-pair RP-HPLC (IP-RP-HPLC). The deep-etched (32.0 μm) silicon micropillar array was fabricated by advanced deep UV lithography and by a dedicated Bosch etch process and then sealed by anodic bonding to a Pyrex glass. The pillar surface was subsequently conditioned hydrophobic. Working in isocratic mode under nonretained conditions, van Deemter curves of dsDNA and coumarin were established to assess the performance of the micropillar array column, resulting in plate heights of only a few micrometers. Working in gradient mode, separations of dsDNA fragments were evaluated. The relevant gradient operation parameters were studied to understand their influence on dsDNA separations. The correlation between DNA length and retention was measured and theoretically described in a length range of 50-500 bp, promising for the determination of DNA of an unknown length. Finally, a separation example demonstrated the excellent separation power of on-chip IP-RP chromatography by achieving a large operation range of DNA length (10-300 bp) with a 5-bp difference among 11 dsDNA fragments.

Quantification of lactose using ion-pair RP-HPLC during enzymatic lactose hydrolysis of skim milk

The correct labelling of dairy foods as “lactose-free” requires a suitably sensitive and valid analytical method for the quantification of lactose in complex food matrices. Thus, an ion-pair RP-HPLC method for the simultaneous determination of lactose, glucose and galactose in original skim milk was investigated. The samples derived from an enzymatic lactose hydrolysis approach (0.5L) using the commercial β-galactosidase Godo-YNL2. After derivatisation with p-aminobenzoic acid and sodium cyanoborohydride, the samples were injected on a RP-C18 column. Tetrabutylammonium hydrogen sulphate was used as the ion-pair reagent in the eluent system. The sugars were quantified using photometric- (UV; 303nm) and fluorescence-detection (λex 313nm, λem 358nm). The overall run time was 27min. The limits of detection (LOD) were estimated at 2mgL−1 (UV detection) and at 0.13mgL−1 (fluorescence detection). The limits of quantification were 6mgL−1 (UV detection) and 0.45mgL−1 (fluorescence detection). Thus, this analytical method is suitable for sensitive lactose quantification in milk systems of less than 10mgL−1.

The Effect of ITPA Polymorphisms on the Enzyme Kinetic Properties of Human Erythrocyte Inosine Triphosphatase Toward its Substrates ITP and 6-Thio-ITP

The role of inosine triphosphatase (ITPase) in adverse drug reactions associated with thiopurine therapy is still under heavy debate. Surprisingly, little is known about the way thiopurines are handled by ITPase. We studied the effect of ITPA polymorphisms on the handling of inosine triphosphate (ITP) and thioinosine triphosphate (TITP) to gain more insight into this phenomenon. Human erythrocyte ITPase activity was measured by incubation with ITP using established protocols, and the generated inosine monophosphate (IMP) was measured using ion-pair RP-HPLC. Molecular analysis of the ITPA gene was performed to establish the genotype. Kinetic parameters were established for the two common polymorphisms for both ITP and TITP as substrates using the above mentioned protocol. Both ITP and TITP are substrates for ITPase and their enzyme activities are comparable. Substrate binding is not altered in the different ITPA polymorphisms. It is shown that the velocity of pyrophosphohydrolysis is compromised when the c.94C > A polymorphism is present, both in the heterozygous and in the homozygous state. TITP is handled by ITPase in a similar way as for ITP, which implies that TITP will accumulate in the erythrocytes of patients with an ITPase deficiency, resulting in adverse drug reactions (ADRs) on thiopurine therapy. In carriers of ITPA polymorphisms, the matter is more complex and the development of ADR may depend on additional epigenetic factors rather than on the accumulation of thiopurinenucleotides.

A robust method for speciation, separation and photometric characterization of all [PtCl 6− nBr n] 2− ( n = 0–6) and [PtCl 4− nBr n] 2− ( n = 0–4) complex anions by means of ion-pairing RP-HPLC coupled to ICP-MS/OES, validated by high resolution 195Pt NMR spectroscopy

A robust reversed phase ion-pairing RP-HPLC method has been developed for the unambiguous speciation and quantification of all possible homoleptic and heteroleptic octahedral platinum IV [PtCl 6− n Br n ] 2− ( n = 0–6) as well as the corresponding platinum II [PtCl 4− n Br n ] 2− ( n = 0–4) complex anions using UV/Vis detection. High resolution 195Pt NMR in more concentrated solutions of these Pt II/IV complexes (≥50 mM) served to validate the chromatographic peak assignments, particularly in the case of the possible stereoisomers of Pt II/IV complex anions. By means of IP-RP-HPLC coupled to ICP-MS or ICP-OES it is possible to accurately determine the relative concentrations of all possible Pt II/IV species in these solutions, which allows for the accurate determination of the photometric characteristics ( λ max and ɛ) of all the species in this series, by recording of the UV/Vis absorption spectra of all eluted species, using photo-diode array, and quantification with ICP-MS or ICP-OES. With this method it is readily possible to separate and estimate the concentrations of the various stereoisomers which are present in these solutions at sub-millimolar concentrations, such as cis- and trans-[PtCl 4Br 2] 2−, fac- and mer-[PtCl 3Br 3] 2− and cis- and trans-[PtCl 2Br 4] 2− for Pt IV, and cis- and trans-[PtCl 2Br 2] 2− in the case of Pt II. All mixed halide Pt II and Pt IV species can be separated and quantified in a single IP-RP-HPLC experiment, using the newly obtained photometric molar absorptivities, ɛ, determined herein at given wavelengths.

Simultaneous determination of ascorbic acid, aminothiols, and methionine in biological matrices using ion-pairing RP-HPLC coupled with electrochemical detector

A novel highly sensitive ion-pairing reversed-phase high performance liquid-chromatography/electrochemical detection method for simultaneous determination of l-ascorbic acid, aminothiols, and methionine in biological matrices was developed, optimized, and validated. Reduced forms of the analytes were extracted from the sample matrices with 10% meta-phosphoric acid solution (aqueous). To determine the total vitamin C, the total aminothiols, and the total methionine, samples were treated with tris(2-carboxyethyl)phosphine solution in 0.05% trifluoroacetic acid solution (aqueous) subsequent to deproteination to reduce the oxidized forms of these compounds. Various analytes were separated on a C 18 (250 × 4.6 mm, 5 μm) analytical column using methanol–0.05% trifluoroacetic acid solution (aqueous) (05/95, v/v), containing 0.1 mM 1-octane sulphonic acid as the ion-pairing agent) as the isocratic mobile phase pumped at a flow rate of 1.5 mL min −1 at room temperature. The column eluents were monitored at a voltage of 0.85 V. These analytes were efficiently resolved in less than 20 min using n-acetyl cysteine as the internal standard. The present method was specific for the analysis of these analytes and demonstrated acceptable values for linearity ( r 2 > 0.999 in the range of 0.2–10,000 ng mL −1 for all the analytes), recovery (>96%), precision (%RSD ≤ 2.0), and sensitivity (on column limit of detection: 250–400 fg and limit of quantification: 0.8–1.25 pg), indicating that the proposed method could be efficiently used for determination of these analytes in the context of clinical research.

Development and validation of an ion-pair RP-HPLC method for the determination of oligopeptide-20 in cosmeceuticals

Oligopeptide-20 is a growth-factor mimicking peptide used in cosmeceuticals. This article describes the development and validation of an ion-pair reversed-phase liquid chromatography method that allows, after liquid–liquid extraction, the quantification of oligopeptide-20 in cosmetic creams. Chromatographic separation was achieved on a cyanopropyl Hypersil analytical column (100mm×2.1mm i.d., 5μm particle size), using a mobile phase of acetonitrile–heptafluorobutyric acid (pH=2.5, 9.0mM) (70:30, v/v) containing 0.045% diethylamine at a flow rate of 0.50mLmin−1. Ultraviolet (UV) spectrophotometric detection at 225nm was used. The method had linear calibration curve over the range 1.35–4.95μgmL−1 for oligopeptide-20. The intra- and inter-day RSD values were less than 3.3%, while the relative percentage error, %Er, was less than 1.9. The developed method was applied successfully to the quality control of a cosmetic cream containing 0.003% (w/w) oligopeptide-20.

HPLC Quantification of 4-Nitrophenol and its Conjugated Metabolites from Bile

An isocratic ion pair RP-HPLC method with UV-Vis detection has been developed and validated for simultaneous analysis of 4-nitrophenol (PNP), 4-nitrophenyl β-glucuronide (PNP-G), and 4-nitrophenyl sulfate (PNP-S) in rat bile samples using 4-ethylphenol (ETP) as internal standard. Chromatographic separation was achieved on a C18 column by isocratic elution with a mobile phase consisted of methanol-0.01 M citrate buffer pH 6.2 (47:53 v/v) containing 0.03 M TBAB. The flow rate was 1.0 ml min−1, the detection was affected at 290 nm. Calibration plots were generated over the concentration range 1–100 μM PNP, PNP-G, PNP-S with a common lower limit of quantification of 2.5 μM. Intra- and inter-day precision and repeatability were determined at six different concentrations. Results obtained by application of the method for determination of PNP, PNP-G and PNP-S in bile fractions collected during intestinal perfusion of PNP in hyperglycemic rats are presented.