Survey of peptide quantification methods and comparison of their reproducibility: A case study using oxytocin.

USP's peptide reference standards content is typically determined using an HPLC assay against an external standard for which the purity was determined by a mass balance approach. To explore the use of other analytical methods, the USP Biologics Department conducted a multi-laboratory collaborative study. The study determined the inter-laboratory variability for peptide quantitation using the following methods: HPLC assay, quantitative nuclear magnetic resonance (qNMR) spectroscopy, or amino acid analysis (AAA). The three methods were compared with regard to their suitability for quantitation of the nonapeptide oxytocin. In this study, the HPLC assay method using the same peptide bulk material as the standard showed the lowest inter-lab variability. The coefficient of variation (%CV) was calculated without counting the uncertainty associated with the purity assignment of the standard with mass balance. The proton qNMR method is a direct measurement of the peptide against an internal standard, which is not difficult to perform under common laboratory conditions. Because of the simpler operation and shorter analytical time, qNMR as a primary method for peptide reference standard value assignment deserves further exploration.

Enabling adoption of 2D-NMR for the higher order structure assessment of monoclonal antibody therapeutics.

The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional 1H,15N and 1H,13C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-15N, 20%-13C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.

Validation of a quantitative NMR method for suspected counterfeit products exemplified on determination of benzethonium chloride in grapefruit seed extracts.

A 1H-nuclear magnetic resonance (NMR) spectroscopy method for quantitative determination of benzethonium chloride (BTC) as a constituent of grapefruit seed extract was developed. The method was validated, assessing its specificity, linearity, range, and precision, as well as accuracy, limit of quantification and robustness. The method includes quantification using an internal reference standard, 1,3,5-trimethoxybenzene, and regarded as simple, rapid, and easy to implement. A commercial grapefruit seed extract was studied and the experiments were performed on spectrometers operating at two different fields, 300 and 600 MHz for proton frequencies, the former with a broad band (BB) probe and the latter equipped with both a BB probe and a CryoProbe. The concentration average for the product sample was 78.0, 77.8 and 78.4 mg/ml using the 300 BB probe, the 600MHz BB probe and CryoProbe, respectively. The standard deviation and relative standard deviation (R.S.D., in parenthesis) for the average concentrations was 0.2 (0.3%), 0.3 (0.4%) and 0.3mg/ml (0.4%), respectively.

Use and qualification of primary and secondary standards employed in quantitative ¹H NMR spectroscopy of pharmaceuticals.

Standards are required in quantitative NMR (qNMR) to obtain accurate and precise results. In this study acetanilide was established and used as a primary standard. Six other chemicals were selected as secondary standards: 3,4,5-trichloropyridine, dimethylterephthalate, maleic acid, 3-sulfolene, 1,4-bis(trimethylsilyl)benzene, and 1,3,5-trimethoxybenzene. The secondary standards were quantified using the primary standard acetanilide. A protocol for qualification and periodic checks of these secondary standards was developed, and used for evaluation of the stability of the compounds. Periodic monitoring of purity was performed for several years. The purity was higher than 99% for all secondary standards. All standards maintained the initial purity during the time period of monitoring, with very small variations in purity (0.3-0.4%). The selected secondary standards were shown to be suitable qNMR standards and that periodic requalification of the standards by qNMR ensures reliable analytical results. These standards have been used in our laboratory for compliance testing of pharmaceutical active substances and approved medicinal products as well as for analysis of suspected illegal medicines. In total more than 1000 samples have been tested using both internal and external standardization and examples are given.

A general analytical platform and strategy in search for illegal drugs.

An effective screening procedure to identify and quantify active pharmaceutical substances in suspected illegal medicinal products is described. The analytical platform, consisting of accurate mass determination with liquid chromatography time-of-flight mass spectrometry (LC-QTOF-MS) in combination with nuclear magnetic resonance (NMR) spectroscopy provides an excellent analytical tool to screen for unknowns in medicinal products, food supplements and herbal formulations. This analytical approach has been successfully applied to analyze thousands of samples. The general screening method usually starts with a methanol extraction of tablets/capsules followed by liquid chromatographic separation on a Halo Phenyl-Hexyl column (2.7µm; 100mm×2.1mm) using an acetonitrile/0.1% formic acid gradient as eluent. The accurate mass of peaks of interest was recorded and a search made against an in-house database containing approximately 4200 substances, mostly pharmaceutical compounds. The search could be general or tailored against different classes of compounds. Hits were confirmed by analyzing a reference substance and/or by NMR. Quantification was normally performed with quantitative NMR (qNMR) spectroscopy. Applications for weight-loss substances like sibutramine and orlistat, sexual potency enhancement (PDE-5 inhibitors), and analgesic drugs are presented in this study. We have also identified prostaglandin analogues in eyelash growth serum, exemplified by isopropyl cloprostenate and bimatoprost. For creams and ointments, matrix solid-phase dispersion (MSPD) was found to give a clean extracts with high recovery prior to LC-MS analyses. The structural elucidation of cetilistat, a new weight-loss substance recently found in illegal medicines purchased over the Internet, is also presented.

Survey and qualification of internal standards for quantification by 1H NMR spectroscopy.

In quantitative NMR (qNMR) selection of an appropriate internal standard proves to be crucial. In this study, 25 candidate compounds considered to be potent internal standards were investigated with respect to the ability of providing unique signal chemical shifts, purity, solubility, and ease of use. The (1)H chemical shift (delta) values, assignments, multiplicities and number of protons (for each signal), appropriateness (as to be used as internal standards) in four different deuterated solvents (D(2)O, DMSO-d(6), CD(3)OD, CDCl(3)) were studied. Taking into account the properties of these 25 internal standards, the most versatile eight compounds (2,4,6-triiodophenol, 1,3,5-trichloro-2-nitrobenzene, 3,4,5-trichloropyridine, dimethyl terephthalate, 1,4-dinitrobenzene, 2,3,5-triiodobenzoic acid, maleic acid and fumaric acid) were qualified using both differential scanning calorimetry (DSC) and NMR spectroscopy employing highly pure acetanilide as the reference standard. The data from these two methods were compared as well as utilized in the quality assessment of the compounds as internal standards. Finally, the selected internal standards were tested and evaluated in a real case of quantitative NMR analysis of a paracetamol pharmaceutical product.

Effect of Ca2+ on the 1H NMR chemical shift of the methyl signal of oversulphated chondroitin sulphate, a contaminant in heparin.

The chemical shift of the methyl signal of oversulphated chondroitin sulphate (OSCS) is dependent on the type and concentration of the counterion. When OSCS is present as a contaminant in heparin sodium, the reported methyl 1H chemical shift is 2.15 +/- 0.02 ppm. In this report, a value of 2.18 +/- 0.01 ppm is reported for the OSCS in the presence of Ca2+. The chemical shift of the methyl signal of pure OSCS varies linearly from 2.13 ppm to 2.18 ppm with increasing amounts of Ca2+, until reaching the saturation point of four Ca2+ ions per OSCS disaccharide unit, which contains four sulphate groups (a 1:1 ratio between sulphate groups and Ca2+). This Ca2+ effect can be used for OSCS identification as well as to facilitate quantification.

Adverse effects by artificial grapefruit seed extract products in patients on warfarin therapy.

OBJECTIVE: Grapefruit seed extract (GSE) is promoted as a natural product with antibacterial and antiviral properties. The aim of this study was to investigate the composition of some commercially available GSE products and evaluate their effect in vitro on two cytochrome P450 enzymes, CYP2C9 and CYP3A4. METHODS: A couple on lifelong treatment with warfarin and continuous regular follow-ups took some drops of a GSE product for 3 days. The female patient experienced a minor subcutaneous haematoma 3 days later, and her international normalised ratio (INR) value was 7.9. This was reported to the Swedish Medical Products Agency (MPA) as a spontaneous post-marketing report concerning adverse drug reactions/interactions. The composition of the GSE products was determined by proton and carbon-13 nuclear magnetic resonance spectroscopy (NMR). The inhibitory effect of the GSE products on the cytochrome P450 enzymes was tested in an in vitro baculosome assay. RESULTS: The NMR analysis showed that all three investigated GSE products contained the synthetic preservative benzethonium chloride (BTC) in addition to glycerol and water. No authentic GSE extract was found in any of the three GSE products analysed. Furthermore, BTC was found to be a potent inhibitor of CYP3A4 and CYP2C9 activity in vitro. CONCLUSION: Our results suggest that BTC in the GSE products is responsible for the increase in the INR value in a patient on warfarin treatment.

Characterization of sulfated beta-cyclodextrins and determination of enantiomeric purity of (1 R,2S)-ephedrine by capillary zone electrophoresis.

Matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize two different sulfated beta-cyclodextrins (HS-beta-CD). It was found that the HS-beta-CDs have broad heterogeneity in terms of sulfation degree. The average sulfate contents were in the range of 6 to 8 per CD molecule. Furthermore the sugar moieties of both HS-beta-CDs were sulfated either at position 6 or at positions 2 and 6, but not at position 3. Enantiomeric separation by capillary zone electrophoresis (CZE) using the HS-beta-CDs as chiral selectors showed that these CDs exhibited similar chiral selectivity and resolution of the ephedrine enantiomers. One of the CDs was employed for the enantiomeric purity evaluation of (1R,2S)-ephedrine (or (-)-ephedrine) by capillary zone electrophoresis. Quantification was done by comparison between the corrected peak areas of the minor enantiomer and (-)-ephedrine.