Continuous screening of analytical parameters facilitates efficient development of HPLC methods required for impurity profiling

Developing a robust analytical HPLC–UV method to characterize a drug candidate during an early stage of development is a major challenge when not all impurity standards are available. Here, we report our efforts to devise an efficient strategy for HPLC method development using continuous screening of analytical parameters without impurity standards. This strategy uses small incremental changes in the mobile phase pH and column temperature to trace each impurity on an overlay chromatogram. We tested this method using benzocaine as the active pharmaceutical ingredient (API), and compounds with similar structures to represent unknown impurities. Despite the coelution of peaks, results identified the number of impurities and indicated the starting point and parameter variables of the ensuing optimization step. Further, we demonstrated that the retention time of each peak as a function of mobile phase pH accounts for the apparent pK_a of known and unknown compounds in the presence of an organic solvent. This information is critically important to the selection of a robust pH range for HPLC methods.     

Development and validation of a capillary electrophoresis method with ultraviolet detection for the determination of the related substances in a pharmaceutical compound

A capillary electrophoresis (CE) method was successfully developed to quantify the impurity profile of a new substance of pharmacological interest: LAS 35917. CE method was developed in order to separate the chloromethylated, monomethylated and hydroxylated impurities (molecules with very similar chemical structures) having the three coelution in the reversed-phase LC method initially established. Taking into account the structure of the impurities of LAS 35917, separation by conventional liquid chromatography (LC) methods would be longer and tedious than separation by CE, which is an appropriate and versatile technique giving easier and quicker methods. Among the three potential impurities mentioned of LAS 35917, two are due to the synthesis route of this drug, and the third arises from degradation. These drug-related impurities were separated using a capillary of 56 cm of effective length and 50 microm I.D., a 60 mM tetraborate buffer, at pH 9.2, and a positive voltage of 20 kV. The optimised CE method was preliminary validated with regard to specificity, linearity, limits of detection and quantitation, repeatability and solution stability. The method allows the detection and quantitation of impurities above 0.04 and 0.08% level, respectively. All three related substances were separated, detected and quantified from their parent drug in the analysis of real samples of LAS 35917, stressed or not stressed, with this simple and fast CE method.     

A stability-indicating ultra-performance liquid chromatographic method for estimation of related substances and degradants in paliperidone active pharmaceutical ingredient and its pharmaceutical dosage forms

A simple, linear gradient, rapid, precise and stability-indicating analytical method was developed for the estimation of related substances and degradants of paliperidone API and tablets. The chromatographic separations were achieved using an Acquity ultra-performance liquid chromatograph (BEH 100 mm, 2.1 mm, 1.7 μm C-18 column) employing 0.01 M potassium dihydrogen phosphate buffer (pH 2.0) as mobile phase A and acetonitrile-water (9:1) as mobile phase B. A linear gradient (mobile phase A, mobile phase B in the ratio of 84:16) with a 0.45 mL/min flow rate was chosen. All six impurities were eluted within five minutes of run time. The column temperature was maintained at 25 °C and a detector wavelength of 238 nm was employed. Paliperidone was exposed to thermal, photolytic, hydrolytic and oxidative stress conditions. The stressed samples were analyzed by the proposed method. Considerable degradation of the analyte was observed when it was subjected to oxidative conditions and impurity F was found to be the major degradant. Peak homogeneity data of paliperidone obtained by photodiode array (PDA) detection demonstrated the specificity of the method in the presence of degradants. The method was validated with respect to linearity, precision, accuracy, ruggedness, robustness, limit of detection and limit of quantification.     

Determination of organic acid impurities in lactic acid obtained by fermentation of sugarcane juice

Lactic acid produced by fermentation process mostly contains a number of aliphatic carboxylic acids as impurities. In this work, carboxylic acid impurities in lactic acid samples from a number of sources were determined at ppm levels. A simple HPLC method was developed that utilized a new generation polar embedded reverse phase, 20mM phosphate buffer at pH 2.20 (±0.05) and UV detection at 210 nm. The method enabled quantitative analysis of the above acids in lactic acid matrix. The experimental conditions for column temperature, mobile phase pH and flow rate were optimized. A detailed validation of the method was performed for linearity, precision, accuracy, selectivity, limit of detection (LOD), limit of quantitation (LOQ), ruggedness and repeatability and reproducibility (R&R).     

Capillary electrophoresis-mass spectrometry in impurity profiling of pharmaceutical products

Generally reversed-phase high-performance liquid chromatography (RP-HPLC) methods are extensively applied during quality control of pharmaceutical products. Since capillary electrophoresis (CE) is based on a different separation principle and consequently results in a unique selectivity compared to RP-HPLC, it can advantageously be used as an orthogonal technique. CE equipped with a mass spectrometer detector provides even more information that can be helpful for identification and structural elucidation purposes. CE-MS was recently implemented in the method development approach to support impurity profiling of pharmaceutical products. In this paper the application of CE-electrospray ionization (ESI)-MS/MS to the impurity profiling of galantamine hydrobromide in stressed Reminyl Extended Release (ER) capsules is discussed. Reminyl ER samples were stressed at different storing conditions. The impurity profile of these samples was compared with the current RP-HPLC and chiral CE method, but also with CE-ESI-MS/MS. The combination of these three methods provided valuable data that allowed understanding comprehensively the impurity profile of these samples. Two impurities were detected at concentrations lower than 0.05%, which did not occur in nonstressed samples. Chromatographic data and the fragmentation patterns of galantamine and related compounds were also examined for identification of these two degradation products.     

Validation of a Stability Indicating LC Method for Amiodarone HCL and Related Substances

A simple, rapid and sensitive isocratic high performance liquid chromatographic (HPLC) method has been developed for the estimation of purity and quantitative determination of Amiodarone HCl active pharmaceutical ingredient (API).The method describes a quantitative estimation of five process related impurities of Amiodarone HCl with a resolution of more than or near to 3.0 between each impurity. These five known related substances are estimated by a simple, rapid and accurate reverse phase isocratic HPLC method. The method has been validated for the determination of assay and related substances in Amiodarone HCl API, using a C₈ column. The elution is carried out using a mobile phase consisting of water-methanol-acetic acid with a pH 5.8. For the quantitative determination of these relative substances, a relative response factors have been determined for all five related substances with respect to Amiodarone HCl. The precision (system precision, method precision and intermediated precision) is demonstrated for both the assay as well as related substances on six independent sample preparations. Accuracy of the method (recovery) is demonstrated for both Amiodarone and each of the five related substances. Specificity of the method is demonstrated by forced degradation study of Amiodarone HCl API under various stress conditions. The method is found to be stability indicating and useful for the analysis of assay and related substances of Amiodarone HCl API in a routine quality control laboratory and for the stability studies of drug substance.     

Analysis of pharmaceutical impurities using multi‐heartcutting 2D LC coupled with UV‐charged aerosol MS detection

To overcome challenges in HPLC impurity analysis of pharmaceuticals, we developed an automated online multi‐heartcutting 2D HPLC system with hyphenated UV‐charged aerosol MS detection. The first dimension has a primary column and the second dimension has six orthogonal columns to enhance flexibility and selectivity. The two dimensions were interfaced by a pair of switching valves equipped with six trapping loops that allow multi‐heartcutting of peaks of interest in the first dimension and also allow “peak parking.” The hyphenated UV‐charged aerosol MS detection provides comprehensive detection for compounds with and without UV chromophores, organics, and inorganics. It also provides structural information for impurity identification. A hidden degradation product that co‐eluted with the drug main peak was revealed by RP × RP separation and thus enabled the stability‐indicating method development. A poorly retained polar component with no UV chromophores was analyzed by RP × hydrophilic interaction liquid chromatography separation with charged aerosol detection. Furthermore, using this system, the structures of low‐level impurities separated by a method using nonvolatile phosphate buffer were identified and tracked by MS in the second dimension.     

Quantification and validation of HPLC‐UV and LC‐MS assays for therapeutic drug monitoring of ertapenem in human plasma

Rapid and simple HPLC‐UV and LC‐MS methods were developed and validated for the quantification of ertapenem (Invanz?) in human plasma. Ertapenem is a unique drug in that current dosing recommendations call for a 1 g dose for normal renal function patients, despite body weight. These assays, which involve a protein precipitation followed by liquid–liquid extraction, allow for fast therapeutic drug monitoring of ertapenem in patients, which is especially useful in special populations. Both methods were sufficient to baseline resolve meropenem (internal standard) and ertapenem, and were validated over 3 days using a six‐point calibration curve (0.5–50 µg/mL). Validation was collected using four different points on the calibrations curve yielding acceptable precision (<15% inter‐day and intra‐day; <20% for lower limit of quantitation, LLOQ) as well as accuracy (<15% inter‐day and intra‐day; <20% for LLOQ). The lower limit of detection (LOD) was determined to be 0.1 and 0.05 µg/mL for the HPLC‐UV and LC‐MS methods, respectively. The developed HPLC‐UV and LC‐MS methods for ertapenem quantification are fast, accurate and reproducible over the calibration range and can be used to determine ertapenem plasma concentrations for monitoring clinical efficacy. Copyright © 2012 John Wiley & Sons, Ltd.     

Development and validation of a capillary electrophoresis method for the characterization of protegrin IB-367.

A capillary electrophoresis (CE) method was developed to characterize protegrin IB-367, an antimicrobial peptide being developed for the treatment of oral mucositis and for other topical applications. The electrophoretic purity and levels of potential impurities/degradation products of IB-367 drug substance are determined by CE using area normalization. Electrophoresis parameters were optimized to allow optimal resolution, reproducibility and minimal analysis time. The separation and resolution between this polycationic peptide and truncated analogs determined by the CE method was much greater than those by the HPLC methods. In addition, the CE methods separates the potential impurities/degradation products from each other while the HPLC methods failed to resolve them. The CE method was validated in the aspects of accuracy, precision, linearity, range, limit of detection, limit of quantitation, specificity, system suitability and robustness. An internal standard was used for the quantitation purpose. The selection criteria of the internal standard as well as the method validation results are presented. The truncated peptide analogs were used to demonstrate the specificity of the method. These analogs were also used to evaluate the limit of quantitation of potential impurities. The relative response factors of these analogs were assessed to determine area normalization feasibility. System suitability tests were established.     

Analysis of potential genotoxic impurities in pharmaceuticals by two-dimensional gas chromatography with Deans switching and independent column temperature control using a low-thermal-mass oven module

The analysis of potential genotoxic impurities (PGIs) in active pharmaceutical ingredients (APIs) is a challenging task. The target limit of detection for a PGI in an API is typically 1 ppm (1 μg/g API). This is about 500 times lower than for classical impurity analysis. Consequently, analytical methods for trace analysis, mostly in combination with MS detection, need to be applied for the qualitative and quantitative determination of these impurities. A two-dimensional capillary GC method is presented that can be used for the determination of some target PGIs. A concentrated solution of the API sample is directly introduced in the GC-MS system, using an apolar column for first-dimension separation. The fraction (heart-cut) containing the PGIs is transferred to a second capillary column, installed in a low-thermal-mass oven (LTM). The LTM focuses the heart-cut(s) and allows independent temperature-programmed analysis with a polar second-dimension column. The API, solvent, and derivatization agents are not introduced in the second column or in the MS detector, avoiding contamination, column degradation, and target analyte peak detection/integration issues. The performance of this set-up is illustrated by the analysis of some Michael-reactive acceptor PGIs and haloalcohols in carbamazepine as test matrix. Excellent reproducibility (<10% RSD) at the low parts per million level and low detection limits (<1 ppm) were obtained.     

Drug impurity profiling strategies

A general scheme is set up for the estimation of the impurity profile of bulk drug substances by the complex use of chromatographic, spectroscopic and hyphenated techniques. Several examples are presented as illustrations to the scheme from the authors' laboratory involving the use of chromatographic methods such as thin-layer-(TLC), gas-(GC), analytical and preparative high-performance liquid chromatography (HPLC), spectroscopic methods such as mass spectrometry (MS) and NMR spectroscopy as well as hyphenated techniques (HPLC/diode-array UV, GC/MS and HPLC/MS). In addition to summarizing earlier work, new examples are also presented: identification of an impurity (propyl 4-[diethylcarbamoyl(methoxy)]-3-methoxy phenylglyoxylate, II) in propanidid (I) and two unsaturated impurities in allylstrenol (VII) by GC/MS and HPLC/diode-array UV as well as estimation of the impurity profile of mazipredone (III) by HPLC/MS and HPLC/diode-array UV.     

Mass spectrometric and gas and high-performance liquid chromatographic behaviour of an impurity in 2,5-hexanedione

The analysis of 2,5-hexanedione, a metabolic compound of several industrial solvents, is normally carried out using gas chromatographic (GC) or GC-mass spectrometric (MS) techniques. This work, with the aim of verifying the possibility of determining the diketone by means of a high-performance liquid chromatographic (HPLC) method with UV detection, illustrates the importance of the choice of a 2,5-hexanedione standard for the determination of the diketone response factor. In some commercial diketone samples the presence of an impurity, which may interfere with the analysis of the target analyte, was ascertained. This impurity showed GC and HPLC behaviour similar to that of 2,5-hexanedione, but gave a very different UV response. This impurity was identified as 3-methylcyclopent-2-enone by means of MS, GC-MS, HPLC-photodiode-array detection, IR and UV spectrometry. The structure was confirmed by comparing the chromatographic, mass and ultraviolet data of the unknown compound with those of a synthesized reference sample. The well known difficulty in determining 2,5-hexanedione by HPLC with UV detection was reconfirmed owing to its low molar absorptivity.     

Impurity profile of macitentan in tablet dosage form using a stability-indicating high performance liquid chromatography method and forced degradation study

Macitentan (MAC) is a pulmonary arterial hypertension (PAH) drug marketed as a tablet and often has stability issues in the final dosage form. Quantitative determination of MAC and its associated impurities in tablet dosage form has not been previously reported. This study quantified impurities present in Macitentan tablets using a binary solvent-based gradient elution method using reversed phase-high performance liquid chromatography. The developed method was validated per International Conference on Harmonization (ICH) guidelines and the drug product was subjected to forced degradation studies to evaluate stability. The developed method efficiently separated the drug and impurities (48 min) without interference from solvents, excipients, or other impurities. The developed method met all guidelines in all characteristics with recoveries ranging from 85%-115%, linearity with r² ≥ 0.9966, and substantial robustness. The stability-indicating nature of the method was evaluated using stressed conditions (hydrolysis:1 N HCl at 80℃/15 min; 1 N NaOH at 25℃/45 min; humidity stress (90% relative humidity) at 25℃ for 24 h, oxidation:at 6% (v/v) H₂O₂, 80℃/15 min, thermolysis:at 105℃/16 h and photolysis:UV light at 200 Wh/m²; Fluorescent light at 1.2 million luxh). Forced degradation experiments showed that the developed method was effective for impurity profiling. All stressed samples were assayed and mass balance was>96%. Forced degradation results indicated that MAC tablets were sensitive to hydrolysis (acid and alkali) and thermal conditions. The developed method is suitable for both assay and impurity determination, which is applicable to the pharmaceutical industry.     

Identification and characterization of the process‐related impurities in fasudil hydrochloride by hyphenated techniques using a quality by design approach

Following the underlying principles of quality by design mentioned in the ICH Q8 guidance, systematic approaches for the control of process‐related impurities have been taken in the manufacturing process of fasudil hydrochloride, a potent Rho‐kinase inhibitor and vasodilator. Three related impurities were found in fasudil hydrochloride lab samples by a newly developed RP‐HPLC with volatile mobile phase gradient elution and UV detection method. The elemental compositions of the impurities were determined by positive ESI high‐resolution TOF‐MS analysis of their [M + H]⁺ ions and their structures were identified through the elucidation of the product mass spectra obtained by a triple quadrupole mass spectrometer. The key impurity was further verified through synthesis and organic spectroscopy including NMR and IR spectroscopy. The origins of these impurities were located and the effective approaches to eliminate them were proposed based on the redesign of the synthetic conditions. The results obtained are important for quality control in the manufacture of fasudil hydrochloride bulk drug substance and injection.     

Determination of relative response factors of impurities in paclitaxel with high performance liquid chromatography equipped with ultraviolet and charged aerosol detectors

A case study was conducted to determine the relative response factors (RRFs) of paclitaxel-related impurities by high performance liquid chromatography (HPLC) equipped with an ultraviolet (UV) detector and charged aerosol detector (CAD) in tandem. The peak response using CAD was independent of analyte structure in an isocratic analysis for this application. After a sample containing known and unknown impurities was analyzed with HPLC-UV-CAD, an empirical approach was developed to calculate the RRFs for all impurities. The RRFs of known impurities were also determined by linear calibration curves. For known impurities, the RRFs values determined with two approaches are comparable. The new approach is effective yet simpler to determine the RRFs for unknown impurities or degradation products since the need for obtaining authentic pure materials was eliminated.     

Development and validation of a HPLC method for 4,7-phenanthroline-5,6-dione I and identification of its major impurity by HPLC-MS-APCI

In this study, the development and validation of an analytical method for the assay of 4,7-phenanthroline-5,6-dione I (dione I) using high-performance liquid chromatography (HPLC) and the determination of its synthetic impurities by employing the method in HPLC-mass spectrometry with atmospheric-pressure chemical ionization and photodiode-array UV detection is reported. The results show that dione I is eluted as a spectrally pure peak resolved from its impurities. 5-Bromo,4-7-phenanthroline is identified as the main impurity. This is supported by elemental analysis of the dione I, which demonstrated the presence of bromine. Validation parameters such as specificity and selectivity, linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ), ruggedness, stability, and system suitability, which are evaluated for this method. The LOD and LOQ are 2.0 microg/mL and 50 microg/mL with a 0.50% relative standard deviation (%RSD), respectively. The calibration curves showed good linearity over the concentration range of 0.05-1.50 mg/mL. The correlation coefficient is > 0.9991 in each case. The %RSD values for intra- and interday precision studies are < 0.40%.     

Performance evaluation of evaporative light scattering detection and charged aerosol detection in reversed phase liquid chromatography

In pharmaceutical industry ultraviolet (UV) detection is often used as the preferred detection technique in HPLC analysis since most pharmaceutical compounds possess a UV-absorbing chromophore. However, in case the active pharmaceutical ingredient (API) does not have a UV-absorbing chromophore, or if some of the impurities present lack a chromophore, they will not be detected in routine HPLC analysis employing only a UV detector and alternative detection schemes have to be used. Refractive index detection or mass spectroscopy (MS) can be used but these detectors have their intrinsic weaknesses, such as lack of sensitivity or high cost. With the appearance of semi-universal techniques such as evaporative light scattering detection (ELSD), and more recent, charged aerosol detection (CAD), detection of non-UV-absorbing compounds became feasible without having to resort to such complex or costly detection methods. This paper evaluates the different performance characteristics such as sensitivity, linearity, accuracy and precision of both the ELSD and CAD detector coupled to HPLC. One disadvantage of this type of detector is the non-linear response behaviour which makes direct linear regression for making calibration curves inaccurate.     

Determination of enantiomeric excess by capillary electrophoresis

Capillary electrophoresis (CE) is becoming an established method for the determination of chiral trace impurities. This paper provides an overview of the state of the art of CE for such determinations. Detection limits of 0.1% impurity is widely accepted as a minimum requirement for chiral trace impurity determinations. This can be relatively easily achieved with CE. However, determination of lower concentrations requires careful optimization of the separation system. Four factors that are of particular significance for trace enantiomeric determinations: resolution, limit of detection, linear range and type of detection, are discussed. Further, the advantages and disadvantages of derivatization in this context are treated as well as the separation approach, ie., direct chiral separation or separation after the formation of diastereomers. It is concluded that the limit of impurity detection can be about 0.05% when UV detection is employed. Using laser-induced fluorescence detection, a quantitative determination at the 0.005% level is often possible.     

Profiling of impurities in illicit methamphetamine by high-performance liquid chromatography and capillary electrochromatography

High performance liquid chromatography (HPLC) with photodiode array (PDA) UV and fluorescence (FL) detection, and capillary electrochromatography (CEC) with laser-induced fluorescence (LIF) detection were investigated for the analysis of acidic extracts derived from illicit methamphetamine. These compounds include major impurities from the hydriodic acid/red phosphorous reduction method, i.e., 1,3-dimethyl-2-phenylnaphthalene and 1-benzyl-3-methylnaphthalene, and other trace-level, structurally related impurities. For certain of these solutes, HPLC with conventional FL detection gave at least a 60× increase in sensitivity over UV detection. In addition, other highly fluorescent impurities were detected in methamphetamine produced via four other synthetic routes. The use of a rapid scanning FL detector (with acquisition of “on the fly” excitation or emission) provided structural information and gave “optimum” excitation and emission detection wavelengths. CEC with LIF detection using UV laser excitation provided greatly improved chromatography over HPLC, with good detection limits in the low ng/ml range. Both methodologies provide good run-to-run repeatability, and have the capability to distinguish between samples.     

Isolation and characterization of degradation products of moxidectin using LC, LTQ FT-MS, H/D exchange and NMR

This study aimed to evaluate the degradation profile and pathways, and identify unknown impurities of moxidectin under stress conditions. During the experiments, moxidectin samples were stressed using acid, alkali, heat and oxidation, and chromatographic profiles were compared with known impurities given in European Pharmacopeia (EP) monograph. Moxidectin has shown good stability under heat, while reaction with alkali produced 2-epi and ?2,3 isomers (impurities D and E in EP) by characteristic reactions of the oxahydrindene (hexahydrobenzofuran) portion of the macrocyclic lactone. Two new, previously unreported, unknown degradation products, i.e. impurity 1 and impurity 2, detected after acid hydrolysis of moxidectin (impurity 2 was also observed to a lesser extent after oxidation), were isolated from sample matrices and identified using liquid chromatography, NMR, high-resolution FT-ICR MS, and hydrogen/deuterium exchange studies. FTMS analysis showed accurate mass of molecular ion peaks for moxidectin at m/z 640.38412, impurity 1 at m/z 656.37952 and impurity 2 at m/z 611.35684, giving rise to daughter ions traceable up to the seventh levels of MSⁿ experiments and supporting the proposed structures. Both unknown impurities along with moxidectin were fully characterized by ¹H, ¹³C, 1D HMBC and 2D (NOESY, COSY and HSQC) NMR experiments. The interpretation of experimental data positively identified impurity 1 as 3,4-epoxy-moxidectin and impurity 2 as 23-keto-nemadectin. The identification of new impurities and correlation of their chromatographic profiles with the EP method is very useful to establish the stability profile of moxidectin and its preparations, as well as add value to the forthcoming moxidectin finished product European Pharmacopeia monographs.