Binary Mobile Phase Research Articles

EFFECT OF NON-AQUEOUS AND BUFFERED MOBILE-PHASE COMPOSITION ON THE RETENTION OF IVABRADINE

□ The chromatographic behavior of ivabradine have been examined using Kieselgel 60 F254, 60 W F254S, and RP-18 W F254S high performance thin layer chromatography (HPTLC) plates as stationary phases. Mobile phases were wide-ranging mixtures (from 0%to 100%, v/v)—non-aqueous and buffered binary mobile phases. Chromatographic retention data and possible retention mechanism were discussed. The electrostatic interactions operating in these chromatographic systems were analyzed by studying the relationship between the log (1/RF ) values and the molecular polarizabilities of solvents as well as the thermodynamic dependence has been shown. Enthalpy and entropy results were calculated from regression coefficients (a, b) and Van't Hoff plot. Gibbs free energy was calculated in accordance with the definition. The influence of the molecular polarizability of the mobile phase on the retention of ivabradine was also examined.

Separation and identification of lipid classes by normal phase LC‐ESI/MS/MS on a cyanopropyl column

In order to establish a versatile and convenient method for the analysis of lipids, electrospray ionization tandem mass spectrometry (ESI‐MS/MS) was applied to a HPLC separation on a cyanopropyl‐bonded stationary phase. A binary gradient mobile phase system consisting of hexane, toluene, methanol and a stable electrospray yielding sodium adduct ions could be used to generate specific product ions in MS/MS mode. By applying the LC/ESI‐MS/MS method on an egg yolk sample, 29 different molecular species of phosphatidylethanolamines, phosphatidylcholines, and lysophosphatidylcholines could be detected within 25 min.

An LC/MS/MS method for stable isotope dilution studies of β-carotene bioavailability, bioconversion, and vitamin A status in humans

Isotope dilution is currently the most accurate technique in humans to determine vitamin A status and bioavailability/bioconversion of provitamin A carotenoids such as β-carotene. However, limits of MS detection, coupled with extensive isolation procedures, have hindered investigations of physiologically-relevant doses of stable isotopes in large intervention trials. Here, a sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) analytical method was developed to study the plasma response from coadministered oral doses of 2 mg [(13)C10]β-carotene and 1 mg [(13)C10]retinyl acetate in human subjects over a 2 week period. A reverse phase C18 column and binary mobile phase solvent system separated β-carotene, retinol, retinyl acetate, retinyl linoleate, retinyl palmitate/retinyl oleate, and retinyl stearate within a 7 min run time. Selected reaction monitoring of analytes was performed under atmospheric pressure chemical ionization in positive mode at m/z 537→321 and m/z 269→93 for respective [(12)C]β-carotene and [(12)C] retinoids; m/z 547→330 and m/z 274→98 for [(13)C10]β-carotene and [(13)C5] cleavage products; and m/z 279→100 for metabolites of [(13)C10]retinyl acetate. A single one-phase solvent extraction, with no saponification or purification steps, left retinyl esters intact for determination of intestinally-derived retinol in chylomicrons versus retinol from the liver bound to retinol binding protein. Coadministration of [(13)C10]retinyl acetate with [(13)C10]β-carotene not only acts as a reference dose for inter-individual variations in absorption and chylomicron clearance rates, but also allows for simultaneous determination of an individual's vitamin A status.

Ultraperformance Liquid Chromatography–Mass Spectrometric Method for Determination of Ampicillin and Characterization of Its Forced Degradation Products

In the current study, a rapid and sensitive UPLC-MS method has been developed for the quantitative analysis of ampicillin in pure and pharmaceutical formulations. Forced degradation analysis was performed and the stress degradation product thus obtained was characterized by mass spectrometry. The chromatographic separation was carried out using BEH C18 column (100 × 2.1 mm, 1.7 µm particle size) using a binary mobile phase mixture of 0.001% acetic acid in water and methanol (30:70). The flow rate was set at 0.3 mL min(-1). The total chromatographic analysis time for ampicillin was as short as 1.5 min. The detection and quantitation of the studied drug was carried out using positive electrospray ionization and selected ion reaction modes. The developed method was found to be linear over the concentration range of 0.25-3.0 µg mL(-1). The recovery studies suggest an excellent recovery of the procedure which was found in the range of 99.45-100.90%. The relative standard deviation range of the developed analytical procedure ranged from 1.98 to 2.67% in intraday studies and 2.38-2.98% in case of interday study. The limit of detection and limit of quantitation of the method were found to be 0.016 and 0.049 µg mL(-1), respectively.

Simultaneous determination of 25 illegally added drugs in diet health foods by ultra-high performance liquid chromatography

An analytical method using ultra-high performance liquid chromatography (UPLC) was developed for qualitative and quantitative analysis of 25 illegally added drugs in diet health foods. The diet food samples were extracted using 40 mL methanol by sonication. After centrifugation, the supernatants were separated on a Waters HSS T3 column with gradient elution at a flow rate of 0.3 mL/min, coupling with diode array detection (DAD) in wavelength range from 200 nm to 400 nm. The binary mobile phase was acetonitrile and 10 mmol/L ammonium acetate solution (containing 0.1% formic acid). The correlation coefficient of standard curve for each drug in linearity range was not less than 0. 997, as well as the recoveries of all the drugs in diet health foods were 70.7%-104% with the relative standard deviations (RSDs) of 0.132%-5.03% at three spiked levels. Seventeen diet food samples were tested, in which phenolphthalein was found in three samples and emodin was found in one sample. The method is specific, easy, quick, and suitable for confirmation of the 25 illegally added drugs in diet health foods.

Preparation and application of an emodin-bonded silica gel chromatographic column for high performance liquid chromatography

An emodin-bonded silica gel stationary phase (ESP) for high performance liquid chromatography has been synthesized by the intermediate method. The preparation process was as follows: γ-glycidoxypropyltrimethoxy silane (KH-560) firstly reacted with emodin ligand; then the intermediate was chemically immobilized to the surface of silica gel. Characterization of ESP was carried out with Fourier transform infrared spectroscopy (FTIR), elemental analysis and thermogravimetric analysis. Naphthalene was used as a probe to determine the column efficiency with methanol-water (60:40, v/v) as binary mobile phase at a flow rate of 0. 8 mL/min. In order to elucidate the related separation mechanism, ESP was used to separate a series of neutral, basic and acidic aromatic compounds, as well as a sample of wind medicated oil. The conventional C18 column and phenyl column were also tested under the same chromatographic conditions for comparison. The results showed that the emodin ligand was successfully bonded to the surface of spherical silica gel with a 0.23 mmol/g of bonded amount, and the theoretical plate number of ESP column was about 19,874 N/m. The coupling reagent chains and anthraquinone rings in ESP offered a structural basis for hydrophobic interaction. Meanwhile, the emodin ligands provided π-π or π-ρ charge transfer, hydrogen bonding, dipole-dipole action sites for different analytes. The synergistic reaction of various action sites endowed ESP column special and excellent chromatographic separation selectivity. And a baseline separation of polar compounds such as amines and phenols on ESP was easily achieved by using common and cheap methanol-water mobile phases without buffer salts. The experimental conditions were simple and convenient.

Validated LC-MS/MS method for simultaneous determination of Dasatinib and Sitagliptin in rat plasma and its application to pharmacokinetic study

Dasatinib (DST), a tyrosine kinase inhibitor, is a novel anticancer agent and Sitagliptin (STG) is an antidiabetic agent of dipeptidyl peptidase-IV inhibitor class. A novel, sensitive and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) based method has been developed for simultaneous monitoring of plasma levels of STG and DST in rat plasma. Both analytes and an internal standard (tolbutamide) were chromatographed on YMC-Pack ODS-AM (50 mm × 4.6 mm i.d., 3 μm) using a methanol : 2 mM ammonium acetate binary gradient mobile phase at a flow rate of 1 ml min−1 with a splitter (1 : 1) over a 5 min run time. Detection of analytes was performed on a LC-MS/MS system in multiple reaction monitoring (MRM) mode. The transitions monitored were 488.1 → 401.0, 408.1 → 174.0 and 271.1 → 155.0 for DST, STG and IS, respectively. The method was validated over a concentration range of 5.41–1029.60 ng ml−1 for DST and 5.64–1073.56 ng ml−1 for STG. The lower limit of quantification was 5.41 ng ml−1 and 5.64 ng ml−1 for DST and STG, respectively. Recoveries from spiked controls were >82% for the analytes and the internal standard at all QC levels. The intra- and inter-batch precision and accuracy across four quality control levels met established criteria of US Food and Drug Administration guidelines. This method was successfully applied to monitor the pharmacokinetic profile of both STG and DST in Wistar rats. This method can be applicable for pharmacokinetic drug–drug interaction studies.

Quantification of bioactive marker β-amyrin by validated high-performance thin-layer chromatographic- densitometric method in different species ofMaytenusgrown in Saudi Arabia

A simple and sensitive high-performance thin-layer chromatographic (HPTLC)-densitometric method was developed and validated for quantification of β-amyrin in the crude extracts of two species of Maytenus (Maytenus obscura and Maytenus parviflora) grown in Saudi Arabia. HPTLC-densitometry was performed on glass-backed silica gel 60 F254 TLC plates with the binary mobile phase hexane-ethyl acetate (3:1, v/v). The developed plate was derivatized with p-anisaldehyde, then scanned and quantified densitometrically at 550 nm. The system was found to give a compact spot for β-amyrin at RF value of 0.38 ± 0.01. The method was found to be satisfactory in terms of sensitivity, accuracy, precision, and recovery. The content of β-amyrin was estimated as 0.42% ± 0.01% and 0.88% ± 0.01% w/w in M. obscura and M. parviflora, respectively. The developed HPTLC technique can be very useful for the quantification of β-amyrin present in various medicinal plants.

Quantitative determination of triterpenoid glycosides in Fatsia japonica Decne. & Planch. using high performance liquid chromatography

Fatsia japonica Decne. & Planch. is a triterpenoid glycoside-rich herb with anti-inflammatory activity for the treatment of rheumatoid arthritis. A method for quantitative analysis of the complex triterpenoid glycosides in this medicinal plant has not been established so far. In this study, a high performance liquid chromatography (HPLC) method was developed for simultaneous qualification of 11 glycosides in F. japonica. The analysis was performed on an ODS-2 Hypersil column (250mm×4.6mm, 5μm) with a binary gradient mobile phase of water and acetonitrile. The established HPLC method was validated in terms of linearity, sensitivity, stability, precision, accuracy, and recovery. Results showed that this method had good linearity with R2 at 0.99992–0.99999 in the test range of 0.04–9.00μg/μL. The limit of detection (LOD) and limit of quantification (LOQ) for the standard compounds were 0.013–0.020μg/μL and 0.040–0.060μg/μL. The relative standard deviations (RSDs%) of run variations were 0.83–1.40% for intra-day and 0.84–3.59% for inter-day. The analyzed compounds in the samples were stable for at least 36h, and the spike recoveries of the detected glycosides were 99.67–103.11%. The developed HPLC method was successfully applied for the measurements of the contents of 11 triterpenoid glycoside in different parts of F. japonica. Taken together, the HPLC method newly developed in this study could be used for qualitative and quantitative analysis of the bioactive triterpenoid glycosides in F. japonica and its products.

Tentative Identification of Torulene Cis/trans Geometrical Isomers Isolated from Sporidiobolus pararoseus by High-Performance Liquid Chromatography-Diode Array Detection-Mass Spectrometry and Preparation by Column Chromatography

The major carotenoids (β-carotene, γ-carotene, torulene, and torularhodin) were determined by high-performance liquid chromatography, with torulene present in the largest amount (167.0 μg/g), followed by torularhodin (113.4 μg/g), β-carotene (52.1 μg/g) and γ-carotene (15.4 μg/g). In addition, cis/trans torulene isomers were further identified by developing an HPLC-DAD coupled with an atmospheric-pressure chemical ionization (APCI) MS method, following isolation and purification torulene from crude pigments by column chromatography. A total of 8 torulene geometrical isomers were resolved within 60 min by employing a YMC C30 column and a binary gradient mobile phase consisting of methanol-methyl tert-butyl ether-water, (50:47.5:2.5, v/v/v) (A) and methanol-methyl tert-butyl ether-water, (8:90:2, v/v/v) (B). Geometrical carotenoid isomers behave differently with respect to bioavailability; therefore, it is of great importance to expand our knowledge on their biological roles to determine the appropriate method to separate torulene cis/trans isomers.

Retention Prediction and Hydrophobicity Measurement of Weakly Basic Compounds in Reversed-phase Liquid Chromatography Using Ammonia and Triethylamine as Ion-suppressors

The purpose of this study is to investigate the retention behavior of weakly ionizable basic compounds in ionsuppression reversed-phase liquid chromatography (IS-RPLC) using ammonia or triethylamine as ion-suppressor in a binary hydroorganic mobile phase. The apparent n-octanol-water partition coefficient (Papp) was proposed to adjust the noctanol- water partition coefficient (Po/w) of weakly basic compound. LogPapp was found to have a better linear correlation with logkw, the logarithm of hypothetical retention factor corresponding to neat aqueous fraction of the mobile phase. This straightforward relationship has been applied to effective prediction of retention behavior of weakly basic compounds, and offered a promising medium for direct determination of Po/w data for these solutes. In addition, the relationship between mobile phase pH and retention behavior of weakly basic compounds was studied in this IS-RPLC mode, and the possible reasons for the biphasic effect of pH on retention have been explained. Keywords: Ion-suppressor, n-Octanol-water partition coefficient (Po/w), retention behavior, reversed-phase liquid chromatography, weakly ionizable basic compound.

Uncertainty of antioxidant profiling in complex mixtures using liquid chromatography involving post-column derivatisation

The main goal of this paper is to discuss the problems associated with antioxidant profiling in complex samples using a high-throughput HPLC system coupled with post-column derivatisation reactor. Based on the experimental data reported in the literature, we demonstrated that improper optimisation of temperature and/or pH assay conditions performed using an on-line derivatisation reactor may substantially change the antioxidant peaks ratio of targeted phytochemical compounds. It has been found that despite the relatively high stability of flavonoids at high temperature and under binary mobile phase HPLC conditions, the reaction of target compounds with common ABTS, FCR and DPPH radicals at elevated regions (particularly above 100°C) dramatically changes their antioxidant activity values expressed, for example, as TEAC (Trolox equivalent antioxidant capacity) parameters. In principle, separation and detection processes of antioxidant profiling assay must not significantly affect the antioxidant activity of target compounds. In the case of foods eaten by humans or animals, critical experimental parameters such as pH and temperature concerning interaction of target analytes with derivatisation reagent should be as close as possible to the real physiological values.

On-line two-dimensional capillary strong anion exchange/reversed phase liquid chromatography–tandem mass spectrometry for comprehensive lipid analysis

An on-line two-dimensional liquid chromatography method was developed for comprehensive lipid profiling by coupling strong anion exchange (SAX) and nanoflow reversed-phase liquid chromatography (nRPLC) prior to electrospray ionization-tandem mass spectrometry (2D-SAX/nRPLC–ESI-MS/MS). Lipids can be classified into four different types according to the electrical propensities of the lipids: anionic, weak anionic, neutral polar, and special lipids. In 2D-SAX/nRPLC, various lipids can be fractionated in the first dimension (SAX: 5μm to 100Å, 5.0cm×75μm i.d.) by step elution (methanol and salt solution), followed by the molecular separation of lipids in the second dimension (RP: 3μm to 100Å, 7.0cm×75μm i.d.) with binary gradient LC. Since the elution of lipids from SAX can be achieved with a very small volume of eluent delivered from an autosampler, it can be simply implemented with an LC–ESI-MS instrument for full automation, and the salt step elution, including the two-step injection procedure, can be used for the selective analysis of the desired lipid fraction. For nRPLC–ESI-MS/MS run in either positive or negative ion mode, a common ionization modifier (0.05% ammonium hydroxide with 5mM ammonium formate) was introduced into the binary mobile phase solutions so that 2D-LC–MS could be operated in both ion modes without changing the mobile phase solutions. The developed on-line 2D-SAX/nRPLC–ESI-MS/MS was evaluated with 22 different standard lipids for the optimization of the salt step elution and was applied to a healthy human plasma lipid extract, resulting in the identification of a total of 303 plasma lipids, including 14 different classes.

Reversed-phase thin-layer chromatography technique for the comparison of the lipophilicity of selected non-steroidal anti-inflammatory drugs

The chromatographic behavior of a series of coxibs and oxicams, drugs from a group of non-steroidal anti-inflammatory drugs, was studied by reversed-phase thin-layer chromatography with binary mobile phases containing water and the organic modifiers: methanol, acetone, 1,4-dioxane, acetonitrile and 2-propanol. Linear relationships were obtained between the retention RM values of the compounds and the concentration of organic modifier in the mobile phase. Values of RM0, represent the theoretical RM values at 0% organic solvent in the mobile phase were calculated by extrapolation. These experimental lipophilicity values were correlated with lipophilicity (logP) from databases. The obtained results show that reversed-phase chromatography (experimental parameters) may be a good instrument for analytics in describing the lipophilic nature of investigated compounds as well as the activity.

Retention Mechanism Studies of Selected Amino Acids and Vitamin B6 on HILIC Columns with Evaporative Light Scattering Detection.

The goal of the study was to investigate separation mechanism of selected “essential” amino acids (leucine, isoleucine, threonine, tryptophan, proline, and glycine) and vitamin B6 in hydrophilic interaction liquid chromatography (HILIC) with the evaporative light scattering detection. Chromatographic measurements were made on three different HILIC columns: amide-silica (TSK-gel Amide-80), amino-silica (TSK-gel NH2-100), and cross-linked diol (Luna HILIC). The retention behaviour of the analytes was investigated as a function of different binary hydro-organic mobile phases containing 10–90 % (v/v) acetonitrile. The compounds studied were separated under isocratic and gradient conditions. The best results of tested biologically active compounds separation were obtained on the TSK-gel NH2-100 column. TSK-gel NH2 column showed mixed HILIC–ion-exchange mechanism, the highest separation efficiency and better selectivity and resolution for tested analytes than the other studied column, especially at concentration of water in mobile phase lower than 30 % (v/v). Special attention was dedicated to the study of interactions among the stationary phase, mobile phase and the analytes.

A semi-automatic microextraction in packed sorbent, using a digitally controlled syringe, combined with ultra-high pressure liquid chromatography as a new and ultra-fast approach for the determination of prenylflavonoids in beers

In this work a highly selective and sensitive analytical procedure based on semi-automatic microextraction by packed sorbents (MEPS) technique, using a new digitally controlled syringe (eVol®) combined with ultra-high pressure liquid chromatography (UHPLC), is proposed to determine the prenylated chalcone derived from the hop (Humulus lupulus L.), xanthohumol (XN), and its isomeric flavonone isoxanthohumol (IXN) in beers. Extraction and UHPLC parameters were accurately optimized to achieve the highest recoveries and to enhance the analytical characteristics of the method. Important parameters affecting MEPS performance, namely the type of sorbent material (C2, C8, C18, SIL, and M1), elution solvent system, number of extraction cycles (extract–discard), sample volume, elution volume, and sample pH, were evaluated. The optimal experimental conditions involves the loading of 500μL of sample through a C18 sorbent in a MEPS syringe placed in the semi-automatic eVol® syringe followed by elution using 250μL of acetonitrile (ACN) in a 10 extractions cycle (about 5min for the entire sample preparation step). The obtained extract is directly analyzed in the UHPLC system using a binary mobile phase composed of aqueous 0.1% formic acid (eluent A) and ACN (eluent B) in the gradient elution mode (10min total analysis). Under optimized conditions good results were obtained in terms of linearity within the established concentration range with correlation coefficients (R) values higher than 0.986, with a residual deviation for each calibration point below 12%. The limit of detection (LOD) and limit of quantification (LOQ) obtained were 0.4ngmL−1 and 1.0ngmL−1 for IXN, and 0.9ngmL−1 and 3.0ngmL−1 for XN, respectively. Precision was lower than 4.6% for IXN and 8.4% for XN. Typical recoveries ranged between 67.1% and 99.3% for IXN and between 74.2% and 99.9% for XN, with relative standard deviations %RSD no larger than 8%. The applicability of the proposed analytical procedure in commercial beers, revealed the presence of both target prenylchalcones in all samples being IXN the most abundant with concentration of between 0.126 and 0.200μgmL−1.

Application of statistical experimental design to the optimisation of microextraction by packed sorbent for the analysis of nonsteroidal anti-inflammatory drugs in human urine by ultra-high pressure liquid chromatography

A new approach based on microextraction by packed sorbent (MEPS) and a reversed-phase ultra-high pressure liquid chromatography (UHPLC) method was developed and validated for the determination and quantification of nonsteroidal anti-inflammatory drugs (NSAIDs) (acetylsalicylic acid, ketoprofen, diclofenac, naproxen and ibuprofen) in human urine. The important factors that could influence the extraction were previously screened using the Plackett–Burman design approach. The optimal MEPS extraction conditions were obtained using C18 phase as a sorbent, small sample volume (20μL) and a short time period (approximately 5min) for the entire sample preparation step. The analytes were separated on a core–shell column (Poroshell 120 EC-C18; 100mm×3.0mm; 2.7μm) using a binary mobile phase composed of aqueous 0.1% trifluoroacetic acid and acetonitrile in the gradient elution mode (4.5min of analysis time). The analytical method was fully validated based on linearity, limits of detection (LOD), limits of quantification (LOQ), inter- and intra-day precision and accuracy, and extraction yield. Under optimised conditions, excellent linearity (R2>0.9991), limits of detection (1.07–16.2ngmL−1) and precision (0.503–9.15% RSD) were observed for the target drugs. The average absolute recoveries of the analysed compounds extracted from the urine samples were 89.4–107%. The proposed method was also applied to the analysis of NSAIDs in human urine. The new approach offers an attractive alternative for the analysis of selected drugs from urine samples, providing several advantages including fewer sample preparation steps, faster sample throughput and ease of performance compared to traditional methodologies.

A simple TLC and HPTLC method for separation of selected steroid drugs

Abstract Chromatographic properties of five steroid drugs: cortisone, hydrocortisone, methylprednisolone, prednisolone and norgestrel have been studied by normal-, reversed-phase and hydrophilic neutral cyano-bonded silica stationary phase with five binary mobile phases (acetonitrile-water, acetonitrile-DMSO, acetonitrile-methanol, acetone-petroleum ether, acetone-water) in which the concentration of organic modifier was varied from 0 to 100% (v/v). This study reports the optimization of steroid hormones separation. Chromatographic retention data and possible retention mechanisms are discussed. Separation abilities of mobile and stationary phases were studied using the principal component analysis method. The best separation of methylprednisolone and prednisolone is with a chromatographic system included silica gel as stationary phase and mixture of acetonitrile and DMSO (10:90 v/v). These two anti-inflammatory drugs can be fast separated from norgestrel when CN is used as stationary phase and acetone and water (40:60 v/v) as mobile phase. The highest values of the parameter Δ(ΔG°) and alfa for cortisone and hydrocortisone was observed in case of using CN as stationary phase and water-acetonitryle (40:60 v/v) as mobile phase.

DEVELOPMENT AND VALIDATION OF RAPID STABILITY INDICATING HPLC-DETERMINATIONS OF ANTIEPILEPTIC DRUGS PHENOBARBITAL IN SUPPOSITORIES AND PHENYTOIN IN CAPSULES AS WELL AS IN URINE SAMPLE

Two simple, rapid, accurate, and stability indicating HPLC methods are described for quantitation of Phenobarbital and Phenytoin in bulk powders, dosage forms, or urine. Chromatographic quantitation of Phenobarbital was developed on Chromolith® Performance RP-18e column, using isocratic binary mobile phase of MeOH and H2O (38: 62, V/V) at flow rate of 3 mLmin−1. Determination of Phenytoin was achieved using a conventional RP C-18, applying isocratic binary mobile phase of ACN and H2O (25: 75, V/V) at flow rate of 1 mL min−1. The elution times of Phenobarbital and Phenytoin are 1.397 ± 0.039 and 5.604 ± 0.013 min, respectively. Each method was validated for system suitability, linearity, precision, limits of detection and quantitation, specificity, stability, and robustness. Stability tests were done through exposure of the analyte solutions to four different stress conditions: reflux with 1 N HCl, reflux with 1 N NaOH, reflux with 30% H2O2, and exposure to UV radiation. Limits of detection and quantitation were 0.125 and 0.250 µgmL−1 as well as 0.250 and 0.500 µgmL−1 for Phenobarbital and Phenytoin, respectively. Due to the short separation time of Phenobarbital, the method was applied for dissolution study in presence of beta-cyclodextrin. The proposed methods can be used for routine samples or stability studies.

Determination of heterocyclic amines in braised sauce beef

A simplified and improved analysis method to determine the content of heterocyclic amines (HAs) was developed. Samples were extracted with ethyl acetate, and macrosubstances were removed by centrifugation. The eluent used consisted of methanol/ammonia water (9:1, v/v), and the eluates were directly mixed for further HPLC analysis. A binary mobile phase consisting of 0.05M ammonium acetate–acetic acid and acetonitrile was used for the separation of 12 HAs, and the excitation and emission wavelengths of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) were adjusted to the same 315 and 410nm, respectively. This method was further applied to determine the content of HAs in “Jiang Niu Rou”, namely braised sauce beef. Nine kinds of HAs were found in the samples and the total amounts of HAs ranged from 4.33 to 27.15ng/g. The sterilized samples contained more HAs than un-sterilized samples.