Doping control analysis of insulin and its analogues in equine urine by liquid chromatography-tandem mass spectrometry

Insulin and its analogues have been banned in both human and equine sports owing to their potential for misuse. Insulin administration can increase muscle glycogen by utilising hyperinsulinaemic clamps prior to sports events or during the recovery phases, and increase muscle size by its chalonic action to inhibit protein breakdown. In order to control insulin abuse in equine sports, a method to effectively detect the use of insulins in horses is required. Besides the readily available human insulin and its synthetic analogues, structurally similar insulins from other species can also be used as doping agents. The author's laboratory has previously reported a method for the detection of bovine, porcine and human insulins, as well as the synthetic analogues Humalog (Lispro) and Novolog (Aspart) in equine plasma. This study describes a complementary method for the simultaneous detection of five exogenous insulins and their possible metabolites in equine urine. Insulins and their possible metabolites were isolated from equine urine by immunoaffinity purification, and analysed by nano liquid chromatography-tandem mass spectrometry (LC/MS/MS). Insulin and its analogues were detected and confirmed by comparing their retention times and major product ions. All five insulins (human insulin, Humalog, Novolog, bovine insulin and porcine insulin), which are exogenous in horse, could be detected and confirmed at 0.05ng/mL. This method was successfully applied to confirm the presence of human insulin in urine collected from horses up to 4h after having been administered a single low dose of recombinant human insulin (Humulin R, Eli Lilly). To our knowledge, this is the first identification of exogenous insulin in post-administration horse urine samples.     

Highly sensitive detection of pharmaceutical compounds in biological fluids using capillary electrophoresis coupled with laser-induced native fluorescence

Insulin administration can increase muscle glycogen by utilising hyperinsulinaemic clamps prior to sports events or during the recovery phases, and increase muscle size by its chalonic action to inhibit protein breakdown. In order to control insulin abuse in equine sports, a method to detect effectively the use of insulins in horses would be required. Besides the readily available human insulin and its synthetic analogues, structurally similar insulins from other species can also be used as doping agents. This study describes a method for the simultaneous detection of bovine, porcine and human insulins, as well as the synthetic analogues Humalog (Lilly) and Novolog (Novo Nordisk) in equine plasma. Insulins were isolated from equine plasma by immunoaffinity purification, followed by centrifugal filtration, and analysed by nano-liquid chromatography-tandem mass spectrometry (LC/MS/MS). Insulin and analogues were detected and confirmed by comparing their retention times and major product ions. All five insulins (human insulin, Humalog, Novolog, bovine insulin and porcine insulin), which are exogenous in the horse, could be detected and confirmed at 0.05ng/mL. This method was successful in confirming the presence of human insulin in plasma collected from horses up to 4h after having been administered a single low dose of recombinant human insulin (Humulin R, Eli Lilly). To our knowledge, this is the first identification of exogenous insulin from post-administration horse plasma samples.     

Validation of the normocythemic mice bioassay for the potency evaluation of recombinant human erythropoietin in pharmaceutical formulations

The normocythemic mice bioassay was validated for the potency evaluation of the recombinant human erythropoietin (rhEPO) against the European Pharmacopoeia Biological Reference Preparation for erythropoietin. The bioassays were performed in 8-week-old female BALB/c mice, which received multiple daily injections of standard or sample solutions (3 + 3), for 4 days. The blood sampling was performed 24 h after the last injection and the reticulocytes were counted by automated flow cytometry. Method validation investigated parameters such as linearity, precision, accuracy, specificity, and robustness, giving results within the acceptable range. The dose-response curve was linear in the concentration range of 1-64 international units (IU)/mL, and the value of the determination coefficient (r2) was 0.9708. The bioassay was applied for the potency evaluation of rhEPO pharmaceutical products containing alfa or beta forms, expressed in different cell lines, giving biological potencies within 82.79 and 119.70% of the stated potency. The precision index calculated by the weight for the independent assays was >247. The results demonstrated the validity of the bioassay for the potency assessment of pharmaceutical formulations contributing to ensure the therapeutic efficacy of the biological medicine.     

Determination of Diphenoxylate Hydrochloride and Atropine Sulphate in Tablet Formulations by Reversed - Phase HPLC

Abstract

The official compendial USP method for the determination of Diphenoxylate HCI (DPHCI) and Atropine Sulphate (ATSO₄) involves extensive sample manipulation followed by non-aqueous titration for (DPHCI) and gas chromatography for (ATSO₄). Furthermore, the assay for individual tablets (content uniformity) is not specific. The proposed HPLC methodology offers substantial improvement in sensitivity, specificity and speed. The method provides simultaneous separation with minimum sample manipulation. The total elution time is less than ten minutes. The accuracy of the method was validated by comparing the results with those obtained by applying the USP XX method on commercial tablets. The specificity of the method was confirmed by the results of content uniformity of DPHCI which were more accurate than those obtained by the USP method.     

Determination of the presence of bovine immunoglobulin G in liquid or spray-dried porcine plasma and whole blood by agar gel immunodiffusion

There is currently urgent interest in identifying the species of origin of the components of different animal by-products. In Europe, this interest is expected to increase with authorization of the re-introduction of these proteins into animal feed formulations. The number of validated methods to differentiate the species of origin for most of these products is limited. An easy, inexpensive, and accurate test was developed to determine the cross-contamination of bovine blood or plasma in porcine whole blood and plasma, both before and after spray drying. Agar gel immunodiffusion (AGID), the studied technique, detected the presence of bovine immunoglobulin G (IgG) in porcine plasma and in whole blood at inclusion levels >0.5% (v/v) in all cases. However, detectability was lower in liquid plasma (0.3%, v/v) and in liquid whole blood (0.5%, v/v). No differences were found when cross-contamination was simulated before or after whole blood centrifugation. The method described is reliable and inexpensive, and the samples are easy to prepare. Both minimal laboratory equipment and expertise are required for detection of bovine IgG in porcine blood products at inclusion levels of >0.5% (v/v).     

A biologically active insulin analogue with modification in the A2 position. [2-Valine-A] sheep insulin

The synthesis and biological evaluation of [2-Valine-A] insulin ([Val2-A]insulin) is reported. In this insulin, the isoleucine residue in position A2, invariant in the majority of mammalian insulins, is substituted by valine. The same substitution, along with four others, occurs naturally in the insulin produced by the owl monkey. Owl monkey insulin exhibits ca. 20% of the activity of porcine insulin in in vitro insulin assays using human cells in culture. [Val2-A]insulin displays 20-22% of the activity of bovine insulin in in vitro insulin assays using rat liver plasma membranes or isolated rat adipocytes. We suggest that the substitution of valine for isoleucine at position A2 is responsible for all or most of the diminution in potency of owl monkey insulin relative to porcine insulin. The data are discussed with regard to previous findings with insulin analogues in which isoleucine A2 was replaced with norleucine, glycine and alanine.     

High-performance liquid chromatographic determination of pilocarpine hydrochloride and its degradation products using a beta-cyclodextrin column.

A high-performance liquid chromatographic (HPLC) method for the determination of pilocarpine, isopilocarpine, pilocarpic acid and isopilocarpic acid was developed. A beta-cyclodextrin column achieved the separation in less than 10 min. Baseline resolution of all four compounds permitted the determination of each degradation product in the presence of pilocarpine. The calibration graphs for each compound were linear, and pilocarpine degradation products could be determined without a correction for the ultraviolet detector response using a pilocarpine standard. A comparison of beta-cyclodextrin separation with the USP HPLC method demonstrated similar results for pilocarpine contents in several commercial ophthalmic formulations.     

Determination and confirmation of tulathromycin residues in bovine liver and porcine kidney via their common hydrolytic fragment using high-performance liquid chromatography/tandem mass spectrometry

An accurate, reliable, and reproducible analytical method using HPLC/MS/MS for the determination of tulathromycin residues in bovine liver and porcine kidney via their common hydrolytic fragment (CP-60,300) was developed and validated. Briefly, the method involved an initial acid treatment of intact tissues, which yielded the common fragment (CP-60,300). A portion of the acid hydrolyzate was purified by SPE using a strong cation exchange cartridge. Evaporation of the purified extract was followed by reconstitution in aqueous buffer and analysis by HPLC/MS/MS under isocratic conditions. The developed method provided acceptable sensitivity for determinative surveillance of tulathromycin in porcine kidney and bovine liver with an LOQ of 7.50 and 2.75 microg/g, respectively. The overall recovery and precision of 45 determinations of each tissue were 97.8% (5.3%) for porcine kidney and 96.9% (7.9%) for bovine liver. Accuracy, precision, linearity, specificity, and ruggedness were demonstrated. An HPLC/MS/MS method was also developed for use in these tissues as a confirmatory assay following modifications to the MS detection parameters. The confirmatory method demonstrated acceptable sensitivity for confirmatory evaluation of tulathromycin in porcine kidney and bovine liver at tolerances of 15 and 5.5 microg/g, respectively.     

Separation of human,bovine, and porcine insulins,three very closely related proteins,by micellar electrokinetic chromatography

Human, bovine, and porcine insulins are small proteins with very closely related amino acid sequences, which makes their separation challenging. In this study, we took advantage of the high‐resolution power of CE, and more particularly of micellar electrokinetic chromatography, to separate those biomolecules. Among several surfactants, perfluorooctanoic acid ammonium salt was selected. Then, using a design of experiments approach, the optimal BGE composition was found to consist of 50 mM ammonium acetate pH 9.0, 65 mM perfluorooctanoic acid ammonium salt, and 4% MeOH. The three insulins could be separated within 12 min with a satisfactory resolution. This method could be useful to detect possible counterfeit pharmaceutical formulations. Indeed, it would be easy to determine if human insulin was replaced by bovine or porcine insulin.     

牛胰岛素去折叠过程的高效液相色谱法分析

建立了反相高效液相色谱法动态监测牛胰岛素在二硫苏糖醇存在下去折叠的过程。牛胰岛素在二硫苏糖醇作用下,首先发生构象变化,形成稳定的中间体后进一步断裂分子间的二硫键,形成Ａ链和Ｂ链。去折叠过程通过基质辅助激光解吸附质谱得到了鉴定。     

Development and validation of an improved liquid chromatographic method for the analysis of dirithromycin

The official method for the determination of dirithromycin and related substances in the European Pharmacopoeia (Ph. Eur.) and in the United States Pharmacopeia (USP) is an isocratic liquid chromatographic (LC) method using an ODS column. With this method, the separation of the main component dirithromycin from its epimer is not complete. Moreover, this method suffers sometimes from drift of the baseline and from subsequent quantitation problems. The required resolution is not easy to obtain.

Using an adapted method derived from the one prescribed in the pharmacopoeias, the selectivity of a set of more than 40 reversed-phase columns towards dirithromycin components was investigated. The selection of the most suitable column was achieved by the chromatographic response function (CRF) approach. Several changes were introduced to the method in order to improve the separation and to overcome the baseline drift problem. The resulting method uses a Zorbax Extend column maintained at 30 °C and a mobile phase containing acetonitrile, methanol, 2-propanol, water and a phosphate buffer at pH 7.5. The method allows a good separation of dirithromycin components, which is much better than that obtained with the existing methods. Several impurities of unknown identity are also separated. The method shows good repeatability, linearity and sensitivity, and it is robust. In addition, it proved to be applicable to a wide number of C18 reversed-phase columns.     

Selection of internal standards for quantitative analysis by matrix-assisted laser desorption-ionization (MALDI) time-of-flight mass spectrometry

The selection of an appropriate internal standard (IS) for quantification by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is critical for the successful application of quantitative MALDI. Selection of the IS depends on the chemical similarity of the analyte and IS and the mass separation of the analyte and IS as a function of instrumental peak resolution. For the quantification of bovine insulin, a series of internal standards including horse heart cytochrome C, bovine insulin chain B, des-pentapeptide human insulin, and des-octapeptide porcine insulin was investigated. Des-pentapeptide human insulin was found to be the most appropriate internal standard (relative standard deviation of the standard curve slope = 2.99%, correlation coefficient = 0.988 in the range of 0.5-0.4 μmol/L). Two methods for measuring of the MALDI signal intensity were evaluated, direct peak integration following subtraction of a linear background and non-linear least squares curve fitting. The results obtained with these methods were equivalent.     

High-performance size-exclusion chromatographic determination of the potency of biosynthetic human growth hormone products

A new high-performance size-exclusion chromatography method has been developed for the determination of potency of human growth hormone products. This method has been extensively validated and shown to correlate well with the hypophysectomized rat bioassay which has been used traditionally. The method is much more precise than the traditional bioassay and thus provides more reliable means of producing consistent biosynthetic human growth hormone batches.     

Influence of operating parameters on the preparative gradient elution chromatography of insulins.

The mass-overloaded separation of bovine and porcine insulins has been studied in the reversed-phase gradient elution mode. Strong solute-solute displacement effects have been found, which are related to the efficiency of the column used. Low flow-rates and small particle diameters maximise the displacements, as well as improve the resolution between the parent insulin and its desamido contaminant. The gradient slope did not substantially affect the separation between the parent insulins, but, due to the relative "S" values of the solutes, an increase in gradient slope improved the separation of the parent insulin from its desamido compound. An optimum pore size of 150 A was found for the insulins. Experiments to optimise the loadability were not carried out, but a recovery of 90% at a purity of 99.5% was obtained with a loading of 12 mg/g of porcine insulin.     

Determination of delta 9-tetrahydrocannabinol in pharmaceutical vehicles by high-performance liquid chromatography

A procedure for the determination of delta 9-tetrahydrocannabinol (delta 9-THC) in the presence of its degradation products in pharmaceutical vehicles by high-performance liquid chromatography (HPLC) is described. The method compares favorably with a standard gas-liquid chromatographic procedure used for the analysis of delta 9-THC in sesame oil USP. The HPLC method is suitable for quantitating delta 9-THC in the presence of several pharmaceutical vehicles and excipients including: sesame oil USP, polyvinylpyrrolidone, Emulphor EL620 and Cremophor EL. Extractions are not required and samples require little preparation. Only the addition of an internal standard in an appropriate solvent is necessary before injection. The procedure has been applied to stability studies of delta 9-THC in various pharmaceutical vehicles.     

Analysis of insulin preparations by reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic system is described for the rapid and complete separation of bovine and porcine insulin from their readily formed monodesamido derivatives under isocratic conditions in the presence of the ion-pairing agent cetrimide. The system is suitable for the direct analysis of formulations of insulins of mixed bovine and porcine origin, and gives satisfactory results with a number of readily available commercial packings. Human insulin is not resolved from porcine in this system, but an alternative system allows the complete separation of all three insulins and their monodesamido derivatives, although acceptable peak shapes were obtained only on a limited number of packings.     

Selection of internal standards for quantitative analysis by matrix-assisted laser desorption-ionization (MALDI) time-of-flight mass spectrometry

 The selection of an appropriate internal standard (IS) for quantification by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is critical for the successful application of quantitative MALDI. Selection of the IS depends on the chemical similarity of the analyte and IS and the mass separation of the analyte and IS as a function of instrumental peak resolution. For the quantification of bovine insulin, a series of internal standards including horse heart cytochrome C, bovine insulin chain B, des-pentapeptide human insulin, and des-octapeptide porcine insulin was investigated. Des-pentapeptide human insulin was found to be the most appropriate internal standard (relative standard deviation of the standard curve slope=2.99%, correlation coefficient=0.988 in the range of 0.5–0.4 μmol/L). Two methods for measuring of the MALDI signal intensity were evaluated, direct peak integration following subtraction of a linear background and non-linear least squares curve fitting. The results obtained with these methods were equivalent. Received: 10 November 1995 / Revised: 4 March 1996 / Accepted: 6 March 1996     

Highly sensitive determination and validation of gabapentin in pharmaceutical preparations by HPLC with 4-fluoro-7-nitrobenzofurazan derivatization and fluorescence detection

A sensitive HPLC method with pre-column fluorescence derivatization using 4-Fluoro-7-Nitrobenzofurazan (NBD-F) has been developed for the determination of gabapentin in pharmaceutical preparations. The method is based on the derivatization of gabapentin with (NBD-F) in borate buffer of pH 9.5 to yield a yellow, fluorescent product. The HPLC separation was achieved on a Inertsil C(18) column (250 mm × 4.6 mm) using a mobile phase of methanol water (80:20, v/v) solvent system at 1.2 mL/min flow rate. Mexiletine was used as the internal standard. The fluorometric detector was operated at 458 nm (excitation) and 521 nm (emission). The assay was linear over the concentration range of 5 50 ng/mL. The method was validated for specificity, linearity, limit of detection, limit of quantification, precision, accuracy, robustness. Moreover, the method was found to be sensitive with a low limit of detection (0.85 ng/mL) and limit of quantitation (2.55 ng/mL). The results of the developed procedure for gabapentin content in capsules were compared with those by the official method (USP 32). Statistical analysis by t- and F-tests, showed no significant difference at 95 confidence level between the two proposed methods.     

Single-laboratory validation of a refined AOAC HPLC method 2005.06 for oysters, cockles, and clams in U.K. shellfish

In 2009, a refined HPLC method based on AOAC Official Method 2005.06 was developed and validated for the determination of paralytic shellfish poisoning (PSP) in mussels. A single-laboratory validation study of this method was undertaken here for the analysis of PSP toxins in oysters, cockles, clams, and razor clams. The method was characterized for selectivity, sensitivity, linearity, precision, repeatability, recovery, ruggedness, and uncertainty of measurement. Validation data were utilized to determine method performance characteristics for non-mussel bivalves for all commercially available certified reference toxins, extending the method to dcNEO and dcGTX2,3, where available. A period of parallel testing of oysters, cockles, and clams enabled a comparison of sample toxicities obtained using mouse bioassay (MBA) and HPLC methodologies, although only a very low number of PSP-positive samples were obtained through the United Kingdom official control monitoring program. Results from the MBA and HPLC methods were well-correlated for PSP-negative samples, but the low number of naturally contaminated PSP-positive samples has prevented any comparative statistical assessment of method performance for non-mussels between the two official methods. However, some evidence for potentially significant differences in total saxitoxin equivalents obtained by the two methods in some species has highlighted the need for further comparative testing in non-mussel samples to be conducted prior to implementation of the HPLC method in routine official control monitoring programs.     

Preservation of Biological LH and FSH Activity after Application on HPLC. Comparison between Cation-Exchange and Reverse-Phase High Performance Liquid Chromatography

Abstract

A cation-exchange high-performance liquid chromatography procedure is described for the separation of bioactive lutropin from follitropin in a human urinary gonadotropin standard preparation (1st IRP 70/45). The results clearly demonstrate that recovered protein retained most of its biological activity following the chromatography. Due to a lack of efficient and quick methods for purifying pituitary or urinary gonadotropins, this method could prove to be valuable for this purpose. This method has also been shown to be quick (less than 20 min) and efficient for separation of impurities or degradation products from bovine follitropin with 92% preservation of its biological activity. Our results suggest that ion exchange HPLC procedure will play a powerful role in the isolation of gonadotropins and other biologically active compounds.