Analysis of crude heparin by H NMR,capillary electrophoresis,and strong-anion-exchange-HPLC for contamination by over sulfated chondroitin sulfate

We previously published a strong-anion-exchange-high performance liquid chromatography (SAX-HPLC) method for the detection of the contaminant over sulfated chondroitin sulfate (OSCS) in heparin sodium active pharmaceutical ingredient (API). While APIs have been processed to remove impurities, crude heparins contain insoluble material, chondroitin sulfates, heparan sulfate, and proteins that may interfere with the recovery and measurement of OSCS. We examined 500 MHz ¹H NMR, capillary electrophoresis (CE), and SAX-HPLC to quantify OSCS in crude heparin. Using our standard API protocol on OSCS spiked crude heparin samples; we observed a weight percent LOD and LOQ for the NMR approach of 0.1% and 0.3%, respectively, while the SAX-HPLC method gave values of 0.03% and 0.09%, respectively. CE data was not amenable to quantitative measurement of OSCS in crude heparin. We developed a modified HPLC sample preparation protocol using crude dissolved at the 100 mg/mL level with a 2.5 M NaCl solution. This SAX-HPLC approach gave a weight percent LOD of 0.02% and a LOQ of 0.07% and had better performance characteristics than that of the protocol used for APIs.     

Determination of impurities in heparin by capillary electrophoresis using high molarity phosphate buffers

Oversulfated chondroitin sulfate (OSCS), an impurity found in some porcine intestinal heparin samples was separated from intact heparin by capillary electrophoresis (CE) using a 600 mM phosphate buffer, pH 3.5 as the background electrolyte in a 56 cm × 25 μm i.d. capillary. This method was confirmed in two separate labs, was shown to be linear, reproducible, robust, easy to use and provided the highest resolution and superior limits of detection compared to other available CE methods. Glycosoaminoglycans such as dermatan sulfate and heparan sulfate were separated and quantified as well during a single run. The heparin peak area response correlated well to values obtained using the official assay for biological activity. A high speed, high resolution version of the method was developed using 600 mM lithium phosphate, pH 2.8 in a 21.5 cm × 25 μm i.d. capillary which provided limits of detection for OSCS that were below 0.1%.     

Quality assessment of unfractionated heparin using 1H nuclear magnetic resonance spectroscopy

Due to problems, especially anaphylactoid reactions, raised by impure unfractionated heparin the quality assessment of heparin has to be reconsidered. Neither the USP nor the European Pharmacopoeia are able to guarantee the purity of heparin, i.e., the limitation of oversulfated chondroitin sulfate (OSCS) which was found to be the reason for the allergic adverse effects. In the first run the regulatory authorities ask for (1)H NMR spectroscopic and capillary electrophoretic measurements in order to characterize the impurity profile of heparin. Using an optimized (1)H NMR method the limit of detection for OSCS was found to be 0.1%. In addition, it is possible to reliably quantify both OSCS and dermatan sulfate (DS), the latter being an indicator of poor purification of the unfractionated heparin. Screening of more than 100 heparin samples collected from international markets revealed a high number of samples containing substantial amounts of DS and a number of samples containing OSCS in an amount higher than 0.1%.     

Analysis of pharmaceutical heparins and potential contaminants using 1H-NMR and PAGE

In 2008, heparin (active pharmaceutical ingredient, API) lots were associated with anaphylactoid-type reactions. Oversulfated chondroitin sulfate (OSCS), a semi-synthetic glycosaminoglycan (GAG), was identified as a contaminant and dermatan sulfate (DS) as an impurity. While DS has no known toxicity, OSCS was toxic leading to patient deaths. Heparins, prepared before these adverse reactions, needed to be screened for impurities and contaminants. Heparins were analyzed using high-field ¹H-NMR spectroscopy. Heparinoids were mixed with a pure heparin and analyzed by ¹H-NMR to assess the utility of ¹H-NMR for screening heparin adulterants. Sensitivity of heparinoids to deaminative cleavage, a method widely used to depolymerize heparin, was evaluated with polyacrylamide gel electrophoresis to detect impurities and contaminants, giving limits of detection (LOD) ranging from 0.1% to 5%. Most pharmaceutical heparins prepared between 1941 and 2008 showed no impurities or contaminants. Some contained DS, CS, and sodium acetate impurities. Heparin prepared in 2008 contained OSCS contaminant. Heparin adulterated with heparinoids showed additional peaks in their high-field ¹H-NMR spectra, clearly supporting NMR for monitoring of heparin API with an LOD of 0.5–10%. Most of these heparinoids were stable to nitrous acid treatment suggesting its utility for evaluating impurities and contaminants in heparin API. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4017–4026, 2009     

Recent developments in sensing of oversulfated chondroitin sulfate in heparin. A review

Oversulfated chondroitin sulfate (OSCS), a non-natural sulfated glycosaminoglycan, recognizes as a significant containment in the pharmaceutical heparin, and it could trigger adverse reactions. Chromatography-, electrophoresis-, electrochemistry-, and spectroscopy-related techniques are currently available for accurate and precise analysis of a trace amount of OSCS in heparin. Recently, emerging studies focus on developing colorimetric and fluorescent probes to monitor OSCS containments in heparin. Therefore, this current review aims to describe the sensing principle and procedure of the reported probes that are sensitive and selective toward OSCS in heparin without the interferences of other sulfated glycosaminoglycans. The reported OSCS-specific probes are comprehensively discussed according to the recognition elements of OSCS, including coralyne, AG73 peptides, positively charged tetraphenylethene derivatives, polythiophene polymer, and poly-l-lysine, protamine, superpositively charged green fluorescent proteins, and poly (diallyldimethylammonium chloride). The sensing of OSCS in heparin is generally achieved using, (i) the specific affinity of the recognition element with OSCS and heparin, (ii) heparinase-mediated hydrolysis of heparin, and (iii) OSCS-induced inhibition of heparinase activity. Additionally, coralyne-based DNA probes can detect OSCS in heparin in the presence of Ca²⁺ ions without the assistance of heparinase. This review will pave the way to design another sensing probe towards other sulfated contaminants, like dermatan sulfate.     

Determination of dermatan sulfate and chondroitin sulfate as related substances in heparin by capillary electrophoresis

Capillary electrophoresis (CE) was applied to the quantitation of dermatan sulfate (DS) and chondroitin sulfate (CS) as related substances in sodium heparin. The method is based on the selective digestion of either CS and DS contained in the main drug heparin, by using chondroitinase ABC (specific for both DS and CS) and chondroitinase AC (specific for only CS). The unsaturated disaccharides released after exhaustive digestion, can be separated by CE using a 110 mM phosphate buffer, pH 3.5 as the background electrolyte in a fused silica capillary (64.5 cm × 50 μm i.d.) at 40 °C and −30 kV. Since the level of each disaccharide released upon enzymatic digestion corresponds to its content in the native glycosaminoglycan, the amount of CS and DS was determined by proportion with the released disaccharides. In particular, ΔUA → GalNAc-4S Na₂ and ΔUA → GalNAc-6S Na₂ were selected for quantitation of CS and DS because of their significant response and short migration time (less than 7 min).The method was validated for linearity, accuracy, precision and it showed to be able in detecting selectively, DS and CS at impurity level (LOD 0.01%, w/w). The proposed CE approach was finally applied to real samples. The results obtained were found in excellent correlation with those achieved by the analysis of the same samples using the official USP method based on high performance anion exchange chromatography (HPAEC) with pulsed amperometric detector.     

Combining (1)H NMR spectroscopy and chemometrics to identify heparin samples that may possess dermatan sulfate (DS) impurities or oversulfated chondroitin sulfate (OSCS) contaminants

Heparin is a naturally produced, heterogeneous compound consisting of variably sulfated and acetylated repeating disaccharide units. The structural complexity of heparin complicates efforts to assess the purity of the compound, especially when differentiating between similar glycosaminoglycans. Recently, heparin sodium contaminated with oversulfated chondroitin sulfate A (OSCS) has been associated with a rapid and acute onset of an anaphylactic reaction. In addition, naturally occurring dermatan sulfate (DS) was found to be present in these and other heparin samples as an impurity due to incomplete purification. The present study was undertaken to determine whether chemometric analysis of these NMR spectral data would be useful for discrimination between USP-grade samples of heparin sodium API and those deemed unacceptable based on their levels of DS, OSCS, or both. Several multivariate chemometric methods for clustering and classification were evaluated; specifically, principal components analysis (PCA), partial least squares discriminant analysis (PLS-DA), linear discriminant analysis (LDA), and the k-nearest-neighbor (kNN) method. Data dimension reduction and variable selection techniques, implemented to avoid over-fitting the training set data, markedly improved the performance of the classification models. Under optimal conditions, a perfect classification (100% success rate) was attained on external test sets for the Heparin vs OSCS model. The predictive rates for the Heparin vs DS, Heparin vs [DS+OSCS], and Heparin vs DS vs OSCS models were 89%, 93%, and 90%, respectively. In most cases, misclassifications can be ascribed to the similarity in NMR chemical shifts of heparin and DS. Among the chemometric methods evaluated in this study, we found that the LDA models were superior to the PLS-DA and kNN models for classification. Taken together, the present results demonstrate the utility of chemometric methods when applied in combination with (1)H NMR spectral analysis for evaluating the quality of heparin APIs.     

An assessment of polydispersed species in unfractionated and low molecular weight heparins by diffusion ordered nuclear magnetic resonance spectroscopy method

The primary goal of this project is to extend a ¹H NMR based method, which combines elements of separation on the basis of molecular size with the information specific to ¹H-1D NMR, to the assessment of the heparin contaminant oversulfated chondroitin sulfate (OSCS) and process related impurity dermatan sulfate (DS), and their polydisperse degradation products in samples of unfractionated heparins (UFHs) and low molecular weight heparins (LMWHs) used as the active pharmaceutical ingredients (APIs) in finished pharmaceutical products. The method has been briefly introduced by us in a recent contribution (vide infra). We propose a labelling of the N-acetyl peaks in the ¹H NMR spectra of the UFHs and LMWHs with the parameter D_i, the translational diffusion coefficient available from DOSY NMR. It is shown how DOSY can be applied for screening lots of unfractionated and depolymerised heparins for obtaining molecular size information for heparins and any impurities when using ¹H NMR. The evidence has been presented that title method can be applied as a routine means for assessment of the OSCS and DS contaminants and the polydisperse chemical entities present in the UFHs and LMWHs used as the APIs in heparin pharmaceuticals.     

Effect of Ca on the H 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.     

Capillary electrophoresis determination of glucosamine in nutraceutical formulations after labeling with anthranilic acid and UV detection

A new robust CE method for the determination of the glucosamine (GlcN) content in nutraceutical formulations is described after its derivatization with anthranilic acid (2-aminobenzoic acid, AA). The CE separation of derivatized GlcN with AA was performed on an uncoated fused-silica capillary tube (50 μm I.D.) using an operating pH 7.0 buffer of 150 mM boric acid/50 mM NaH₂PO₄ and UV detection at 214 nm. The method was validated for specificity, linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ). The detector response for GlcN was linear over the selected concentration range from 240 to 2400 pg (40–400 μg/mL) with a correlation coefficient greater than 0.980. The intra- and inter-day variations (CV%) were between 0.5 and 0.9 for migration time, and between 2.8 and 4.3 for peak area, respectively. The LOD and the LOQ of the method were approximately 200 and 500 pg, respectively. The intra- and inter-day accuracy was estimated to range from 2.8% to 5.1%, while the percent recoveries of GlcN in formulations were calculated to be about 100% after simple centrifugation for 10 min, lyophilization and derivatization with AA. The CE method was applied to the determination of GlcN content, in the form of GlcN–hydrochloride or GlcN–sulfate, of several nutraceutical preparations in the presence of other ingredients, i.e. chondroitin sulfate, vitamin C and/or methylsulfonylmethane (MSM) as well as salts and other agents. The quantitative results obtained were in total conformity with the label claims.     

Oversulfated chondroitin sulfate interaction with heparin-binding proteins: New insights into adverse reactions from contaminated heparins

An oversulfated chondroitin sulfate (OSCS) was identified as a contaminant to pharmaceutical heparin and severe anaphylactoid reactions were ascribed to this contaminant. An examination of the biochemistry underlying both the anticoagulant activity and the toxic effects of oversulfated chondroitin sulfate was undertaken. This study demonstrates that the anticoagulant activity of this oversulfated chondroitin sulfate is primarily dependent on heparin cofactor II mediated inhibition of thrombin. Heparin and oversulfated chondroitin sulfate binding to coagulation, kinin-kallikrein and complement proteins were studied by surface plasmon resonance. While oversulfated chondroitin sulfate binds tightly to antithrombin III, unlike heparin, OSCS does not induce antithrombin III to undergo the conformational change required for its inactivation of thrombin and factor Xa. In contrast to heparin, oversulfated chondroitin sulfate tightly binds factor XIIa suggesting a biochemical mechanism for the factor XIIa-based enhancement of vasoactive bradykinin production.     

An efficient separation and method development for the quantifying of two basic impurities of Nicergoline by reversed-phase high performance liquid chromatography using ion-pairing counter ions

A quantification method was developed for the two basic impurities, one of which is also a metabolite, of Nicergoline (NIC), by using reversed-phase high performance liquid chromatography (RP-HPLC) and diode array detector (DAD). One of these compounds,10-methoxy-6-methylergoline-8beta-methanol-5-bromo-3-pyridinecarboxylate (1-DN) is the metabolite as well as the impurity whereas, the other 10-methoxy-1,6-dimethylergoline-8beta-methanol-5-chloro-3-pyridinecarboxylate (5-CN) is only an impurity. The chromatographic column was Phenomenex, Luna, 5 microm, C18 (2), 250 mm x 4.6 mm. Mobile phase was 0.1 M ammonium acetate (NH4Ac) solution containing 4 mM 1-octanesulfonicacid sodium salt (OSASS) and 6 mM tetrabutylammonium hydrogen sulphate (TBAHS) (pH: 5.9)/acetonitrile (ACN) (62:38) for 1-DN and (64:36) for 5-CN. Flow rate was 1.0 mL min-1. The diode array detector was operated at 285 nm, band width: 4 nm. Linearity was obtained in the concentration range of 0.032 x 10-5 to 3.828 x 10-5 M, y = 116.88x + 0.2773 (r2 = 0.99989); the limit of detection (LOD) and limit of quantification (LOQ) were determined as 0.012 x 10-5 and 0.041 x 10-5 M for 1-DN, respectively. Linearity was obtained in the concentration range of 0.034 x 10-5 to 4.092 x 10-5 M, y = 104.24x + 0.7486 (r2 = 0.99996); (LOD) and (LOQ) were determined as 0.014 x 10-5 and 0.046 x 10-5 M for 5-CN, respectively. The recovery was 100.65% for 1-DN and 100.32% for 5-CN. The amount of 1-DN in 30 mg NIC was found as 209.65 microg (0.70%) and the amount of 5-CN in 30 mg NIC was found as 27.62 microg (0.09%).     

Assay of possible economically motivated additives or native impurities levels in heparin by H NMR,SAX-HPLC,and anticoagulation time approaches

New assays have been developed to detect the presence of economically motivated additives (EMAs) and poor manufacturing processes in heparin. Here, selected oversulfated glycosaminoglycans that are possible EMAs to heparin were synthesized and the utility of current ¹H NMR, SAX-HPLC or anticoagulation time protocols were evaluated for the detection of native impurities (chondroitin sulfate A or B (CSA or CSB), or heparan sulfate (HS)), or synthetic contaminants (oversulfated-(OS)-CSA, OS-CSB, OS-HS or OS-Heparin) spiked into heparin sodium active pharmaceutical ingredients (APIs). The range of w/w percent LOD values from the SAX-HPLC analysis for heparin spiked with CSA, CSB, HS, OS-CSA, OS-CSB, OS-HS, OS-Heparin and two partially oversulfated CSA analogs was 0.05–0.12%. The 500 MHz 1D-¹H NMR spectra of heparin spiked with 1.0–10% CSA, CSB, OS-CSA, or OS-CSB showed unique signal pattern changes while the samples spiked with HS, OS-HS, OS-Heparin or partially sulfated CSA were more difficult to identify using NMR data. The ratio of anticoagulation time values obtained with factor Xa and factor IIa were found to remain within USP specifications in the presence of 10% amounts of these impurities or contaminants. In a separate test, using OS-CSA spiked API heparin samples, the factor Xa or factor IIa to USP standard ratio were found to fall below the USP 0.9 specification value in samples spiked at ca. weight percent of 15% or greater of OSCS. We conclude that the SAX-HPLC assay is the most sensitive and robust assay to identify and quantitate possible GAG-based EMAs in heparin.     

高效液相色谱法测定尼莫地平及制剂中光降解产物的含量

目的：建立尼莫地平及制剂中光降解产物含量的测定方法。方法：采用反相高效液相色谱法,Shiseido CAPCELL PAK C18（250mm×4．6mm,5μm）色谱柱,流动相为甲醇-乙腈-水（35：38：27）,流速为1．0ml／min,检测波长为235nm。结果：尼莫地平和杂质A在0．1—4μg／ml的浓度范围内,均与其峰面积呈良好线性（尼莫地平：r=0．999996,n=6;杂质A：r=0．999995,n=6）。精密度试验尼莫地平的RSD为0．1％（n=5）,杂质A的RSD为1．2％（n=5）,尼莫地平的最低检测限为0．02ng,杂质A的定量限为0．2ng。结论：本方法简便、结果准确,能控制尼莫地平及其制剂的质量。     

高效液相色谱法测定硫酸阿托品注射液的含量

目的建立测定硫酸阿托品注射液含量的高效液相色谱（HPLC）法。方法采用Capcell PAK C18色谱柱（250mm×4．6mm,5μm）,柱温35℃,以0．05mol／L磷酸二氢钠溶液（用磷酸调节pH至3．0）-乙腈（82：18）为流动相,流速1．0mL／min,检测波长202nm。结果硫酸阿托品质量浓度在20．62～185．54μg／mL范围内与峰面积线性关系良好（r=1．0000）,检测限为0．41μg,定量限为1．2μg,低、中、高3个质量浓度水平的回收率（n=3）分别为99．58％（RSD=0．96％）,99．60％（RSD=0．53％）,99．51％（RSD=0．41％）。结论HPLC法专属、简便、准确可靠,可作为该制剂的质量控制方法。     

Monitoring the purity of pharmaceutical heparin preparations by high-field 1H-nuclear magnetic resonance spectroscopy

High-field (300 MHz) 1H NMR spectral analyses are reported for various sodium or calcium heparin products available on the Canadian market. Dermatan sulfate (chondroitin sulfate B) was detected as a contaminant in virtually all of these products. Its content varied among the suppliers from less than 1 to 15%, and also over nearly the same range within the groups of heparin preparations of particular suppliers. No correlation was found between in vitro biological activities (potency and anti-factor Xa by the USP tests) and the levels of dermatan sulfate found. Other components, or unlisted constituents, detected in some preparations were paramagnetic metal ions, polyols, and lidocaine.     

Separation and determination of clopidogrel and its impurities by capillary electrophoresis

Clopidogrel bisulphate, an anti-platelet drug, has been separated from its impurities, namely impurity A, B and C by capillary zone electrophoresis (CZE) using uncoated fused-silica capillary (50.0 μm internal diameter, 31.2 cm total length). Four factors affected the separation: buffer concentration, pH of the buffer, concentration of the chiral selector and the applied voltage. Optimization and robustness studies were performed with the aid of reduced central composite experimental design. The buffer used was triethylamine–phosphoric acid and the chosen chiral selector was sulphated β-cyclodextrin (SCD). The best separation was achieved by using 10 mM buffer, pH 2.3, containing 5% (mass/volume (m/v)) SCD. Reversed polarity mode was used with an applied voltage of −12 kV and the capillary temperature was maintained at 20 °C. The method was validated for quantitative determination of the drug. It offered a limit of detection (LOD) of 0.13 μg/ml, a limit of quantitation (LOQ) of 0.4 μg/ml, and a linearity range of 0.4–300 μg/ml. Commercial bulk samples were analyzed using the developed method.     

Hantzsch pre-column derivatization for simultaneous determination of alendronate sodium and its pharmacopoeial related impurity: Comparative study with synchronous fluorometry using fluorescamine

High performance liquid chromatographic (HPLC) method with a pre-column derivatization based on Hantzsch condensation reaction was applied for simultaneous determination of alendronate sodium (ALN) and its main related impurity, 4-Aminobutanoic acid (ABA) at its pharmacopeial limit. The separation of colored condensation products of ALN and ABA were achieved on Agilent Zobrax Eclipse SB-C18 analytical column (250 × 4.6 mm, 5 μm) using a mobile phase composed of acetonitrile–0.1 M acetate buffer, pH 5.0 (15:85, v/v). The flow rate was 1 mL min⁻¹. The detection was carried out at 340 nm using photo-diode array detector. Peak areas were used for the linear regression line in the range of 10–500 and 0.2–40 μg mL⁻¹ for ALN and ABA, respectively. Different conditions for the optimization of the derivatization reactions as well as for the HPLC measurement were studied. The proposed method was validated for linearity, precision, accuracy, specificity and robustness. This method was used to check the purity of ALN in the presence of ABA (related impurity) at the pharmacopeial limit (0.5%). For comparison purpose, another method was proposed which involves synchronous fluorescence measurement after ALN reaction with fluorescamine. In this method, the third derivative synchronous spectra were estimated as peak to peak measurement from 339 to 370 nm for ALN determination with LOD and LOQ of 24 and 73 ng mL⁻¹, respectively, showing very high sensitivity. Both methods have been applied for determination of the alendronate sodium (ALN) in bulk and pharmaceutical preparations without interference of additives in tablets or oral solution.     

电导法测定多硫酸肝素钠中硫酸基含量

目的：建立一种测定多硫酸肝素钠中硫酸基含量的方法：方法：将多硫酸肝素钠通过H^＋型阳离子树脂，用氢氧化钠对其洗脱液进行滴定，通过电导率的变化确定滴定终点，并用氯代十六烷基吡啶对结果进行校正，测定其硫酸基含量。结果：硫酸基的平均加样回收率为100．18％，RSD为3．67％。结论：本法简便准确，重复性较好。     

Determination of amlodipine in pharmaceutical formulations by differential-pulse voltammetry with a glassy carbon electrode

A differential-pulse voltammetric method was developed for the determination of amlodipine based on the oxidation of the dihydropyridine group on the surface of glassy carbon electrode under stationary and rotating conditions. The experiments were conducted in a supporting electrolyte consisting of 0.2 M KCl, 0.1 M phosphate buffer, and 10% (v/v) methanol during investigation of initial potential and pH effects. No adsorption effect was observed on using an initial potential of 0 mV and the supporting electrolyte solution at pH 5.5 under both stationary and rotating conditions. The factor affecting the voltammetric current was diffusional in the range of 200-1000 rpm for rotating, and 2-40 mV s-1 for stationary conditions up to a concentration of 0.04 mg mL-1 amlodipine. The limit of detection (LOD) and the limit of quantitative (LOQ) for the rotating and stationary techniques were found to be 0.004 and 0.0072 mg mL-1 (for S/N = 3.3) and LOQ 0.012 and 0.022 mg mL-1 (for S/N = 10), respectively. The proposed method was applied to the tablets containing amlodipine and according to the statistical evaluations acceptable results were obtained at the 95% probability level.