Development and Validation of a High-Performance Size

Original Research Paper

Acta Chromatographica 29(2017)2, 193–205 DOI: 10.1556/1326.2017.29.2.4

This is an open-access article distributed under the terms of the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium fornon-commercial purposes, provided the original author and source are credited. First published online: July 26, 2016 ISSN 2083-5736 © The Author(s)

Development and Validation of a High-Performance Size-Exclusion

Chromatography Method for Polymer Determination in Cefmetazole Sodium for

Injection

J. ZHONG1,*, Y.C. CHEN2, AND X.Y. LIU3

1Department of Pharmacy, Nanjing Medical University Affiliated Wuxi Second Hospital,

Wuxi 214002, China 2Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai 200040, China 3Department of Chemistry, the Institute for Drug Control of Wuxi, Wuxi 214000, China

*E-mail: [email protected]

Summary. This study aims to develop and validate a high-performance size-exclusion chromatography (HPSEC) method to determine the amount of polymer in cefmetazole sodium for injection and to compare this method with gel chromatography. A Zenix SEC-150 column was used with the mobile phase of phosphate buffer solution (pH 7.0; 0.01 M)–acetonitrile (90:10 v/v) at a flow rate of 0.8 mL min−1 and a detection wavelength of 240 nm. The polymer was quantified by an external standard method with self-control, and the amount was expressed by the percentage of cefmetazole. The HPSEC method was validated for specificity, linearity, and precision. The chromatographic conditions, chromatographic performances, sensitivity, linearity, and precision of the developed HPSEC method and gel chromatography were compared, and both methods were sub-sequently used to determine the amount of polymer from seven batches of samples. The HPSEC method was fully validated. The time of isocratic elution for sample assay was less than 14 min. The results of comparison indicate that the developed HPSEC method was superior to gel chromatography. The Student t test results also showed significant difference in the amount of polymer from the samples obtained by the two methods. Thus, the HPSEC method with two obvious advantages, the superior sensitivity and a shorter analysis time, is more suitable for determination of polymer amount in cefmeta-zole sodium for injection to control the quality of the product. Key Words: HPSEC, gel chromatography, polymer, cefmetazole

Introduction Cefmetazole sodium is a second-generation cephalosporin invented by Dai-ichi Sankyo Company Limited. The drug has a good antibacterial efficacy against Gram-positive and Gram-negative bacteria and is widely used in

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clinical practice [1–4]. The polymer of cefmetazole is easily generated during the production and storage of the material. Polymers of β-lactam antibiotics are important in terms of product quality because they are related to drug anaphylaxis [5, 6]. The strict control of the amount of polymer has been at-tracting considerable attention to decrease the risk of anaphylaxis. In the pharmacopoeia, detection of polymer amount is required in an increasing number of β-lactam antibiotics including related substances, such as cefo-dizime and ceftazidime.

At present, gel chromatography on a Sephadex column is the most widely used method to isolate the polymers of β-lactam antibiotics [7–9]. A previous study examined 12 cephalosporins by using different types of Sephadex gel and found that the solid-phase Sephadex G-10 is the best choice to separate polymers [7]. Another study compared three gel filtration chromatographic methods and revealed that the separation on the Superdex peptide column is superior in determining the amount of amoxicillin poly-mers [8]. Similarly, gel chromatography on a Sephadex G-10 column is used to determine the amount of polymer in cefmetazole sodium [9]. However, gel chromatography has certain disadvantages, such as low sensitivity, long time for sample assay, and tedious chromatographic conditions.

A polymer is a substance with a high molecular weight; the separation and quantification of a polymer using filtration chromatography are more dependent on the solid phase. To develop the method mentioned above, we established a high-performance size-exclusion chromatography (HPSEC) method, which is a novel assay to determine the amount of polymer in cefmetazole sodium; chromatographic column packing was used in a ball-type silica gel, whose surface bonds with hydrophilic groups [10], to replace the Sephadex column.

The standard substance of a polymer must be determined to measure its amount; however, this substance is difficult to obtain and is unstable. Thus, the relative amount instead of the absolute amount of a polymer is used in quality control [11]. An external standard method with self-control, and an area normalization method can be used to quantify the relative amount of a polymer. Compared with the external standard method with self-control, the area normalization method yields results with relatively significant variances. Therefore, the external standard method with self-control is a common choice to quantify the amount of polymer.

In the present study, the developed HPSEC method to determine the amount of polymer in cefmetazole sodium for injection was validated. The polymer structure of cefmetazole was conjectured through its molecular weight, which was detected by mass spectrometry. Meanwhile, the devel-


Development and Validation of an HPSEC Method

195

oped method was compared with gel chromatography, and the results show that HPSEC has advantages in speed, sensitivity, linearity, and precision.

Experimental

Materials and Reagents

Standard cefmetazole (Lot No. 130580-201301, purity 98.4%) was obtained from the National Institutes for Food and Drug Control. Cefmetazole so-dium for injection was obtained from Sichuan Hexin Pharmaceutical Co., Ltd. in China, and high-performance liquid chromatography (HPLC)-grade acetonitrile was purchased from Merck (KGaA, Darmstadt, Germany). All other analytical-grade chemicals and reagents were acquired from Nanjing Chemical Reagent Corp. (Nanjing, China).

Preparation of Solutions

Solutions of standard cefmetazole and cefmetazole sodium for injection were prepared before assaying because the polymer is easily generated during storage of the solutions. In this study, various amounts of standard substance and samples were placed in each 10-mL volumetric flask and then diluted with phosphate buffer solution before detecting.

Apparatus and Chromatographic Conditions for HPSEC

HPLC analysis was performed using an HP1200 LC/DAD system (Agilent Technologies, USA) with a Zenix SEC-150 column (7.8 mm × 300 mm 3 μm, 150 Å Sepax Technologies, USA). The mobile phase was a phosphate buffer solution (pH 7.0; 0.01 M)–acetonitrile (90:10 v/v) at a flow rate of 0.8 mL min−1. The detection wavelength was 240 nm. The column tempera-ture was maintained at 25 °C, and 10 μL was injected into the column.


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Apparatus and Chromatographic Conditions for Polymer Identification

The polymer was identified using an Infinity1290 LC/6530AQ-TOF system (Agilent Technologies, USA) with the same column as the one mentioned above. The mobile phase was ammonium acetate (0.01 M)–acetonitrile (98:2 v/v) at a flow rate of 0.8 mL min−1. The column temperature was maintained at 35 °C, and 1 μL was injected into the column. Liquid chromatogra-phy–electrospray ionization mass spectrometry (LC–ESI–MS) was used with nitrogen to assist nebulization. A quadrupole mass spectrometer equipped with an electrospray ionization source, set with a drying gas (N2) flow of 10 L min−1, nebulizer pressure of 3.5 kV, drying gas temperature of 350 °C, and capillary voltage of 75 V was used in negative-ion selected-ion moni-toring mode. The fragmentor voltage was 120 V, and the collision energy was 20 eV.

The fragmentation pathway of cefmetazole polymer was predicted in accordance with the molecular weight obtained from the above conditions of liquid chromatography–mass spectrometry (LC–MS).

Method of Calculating the Amount of Polymer The polymer peak was ahead of the cefmetazole peak, which is in accor-dance with the mechanism of size-exclusion [12], and the amount of poly-mer was quantified by an external standard method with self-control. These procedures were performed with the assumption that the response of the polymer is identical to that of cefmetazole under the same chromatographic conditions. The calculation formula is as follows: amount of polymer (%) = (fs × Ap / Cx)×100, where fs is the ratio of the concentration and peak area of standard cefmetazole, Ap is the peak area of the polymer, and Cx is the con-centration of the sample. The relative amount, which is commonly used to calculate β-lactam antibiotic polymer [11], was used in the calculation.



197

Assay Validation for HPSEC

Specificity Specificity was evaluated by analyzing the concentration of cefmetazole so-dium for injection at 1 mg mL−1 and analyzing the solution after three kinds of destructive tests: hard light (exposed to 254 nm of UV light for 6 h), high temperature (at 60 °C water bath for 1 h), and a strong oxidizing agent (plus 30% hydrogen peroxide solution 1 mL and placed at room temperature for 0.5 h).

Linearity and LOQ of cefmetazole The standard working solutions of cefmetazole at different concentrations (1, 2, 5, 10, 20, 50, and 100 μg mL−1) and the limit of quantification (LOQ) of cefmetazole were assayed. A calibration curve was constructed by plotting the peak areas of cefmetazole against its concentrations. LOQ for cefmeta-zole was established based on a signal-to-noise (S/N) ratio of 10.

Linearity of polymer

The working solutions of the cefmetazole sodium for injection at concentra-tions of 0.25, 0.5, 0.75, 1, 1.25, 1.5, and 1.75 mg mL−1 were assayed. A cali-bration curve was constructed by plotting the peak areas of the polymer against cefmetazole concentrations.

Precision The precision of the method was assessed in terms of repeatability and in-termediate (inter-day) precision, which was expressed as relative standard deviation (RSD %) of individual measurements for six replicate determina-tions on the same day and six determinations on six different days. The pre-cision values of the quality control (QC) solutions of standard cefmetazole were determined at 1, 10, and 50 μg mL−1, and those for the samples of cefmetazole sodium for injection were determined at 0.25, 1, and 1.5 mg mL−1.


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Stability The stability of the samples was determined by using six replicates at dif-ferent times. The solutions of cefmetazole sodium for injection (1 mg mL−1) were placed at room temperature (25 °C) for 0, 2, 5, 10, and 18 h, respec-tively.

Gel chromatography The gel chromatography by Sephadex G-10 column was run on an Amer-sham AKTA LC high-molecular weight polymer analyzer (Amersham Technologies, USA) [9]. The concentration range of 10–500 μg mL−1 (10, 50, 100, 200, 300, 400, and 500 μg mL−1) was used for evaluating the linearity of cefmetazole, and 2.5–32.5 mg mL−1 (2.5, 7.5, 12.5, 17.5, 22.5, 27.5, or 32.5 mg mL−1) was used for evaluating the linearity of polymer. The QC solu-tions of standard cefmetazole (10, 200, and 400 μg mL−1) and the samples of cefmetazole sodium for injection (2.5, 17.5, and 27.5 mg mL−1) were used to validate the precision of gel chromatography.

Results and Discussion

Assay Validation for HPSEC

Specificity

The chromatograms of cefmetazole sodium for injection, which were ob-tained by HPSEC, are shown in Fig. 1. The method exhibited high specificity and fully achieved baseline separation before and after the destructive tests. The polymer peak was ahead of cefmetazole peak and was identified by mass spectrometry. The sample required less than 14 min of isocratic elution. The retention times were 8.4 min for the polymer and 9.8 min for cefmeta-zole.

Linearity and LOQ of cefmetazole

The calibration curves were linear over the concentration range of 1–100 μg mL−1, with a 0.9999 coefficient of correlation. The equation of the linear regression line was y = 13.898x + 1.1075, and the LOQ of cefmetazole was 1.0 ng mL−1.



199

Fig. 1. Chromatograms of specificity by HPSEC (1 — polymer, 2 — cefmetazole):

(a) destroyed by light, (b) destroyed by heat, (c) destroyed by oxidation, and (d) sample

Linearity of polymer The calibration curves were linear over the concentration range of 0.25–1.75 mg mL−1, with a 0.9997 coefficient of correlation. The equation of the linear regression line was y = 69.314x − 1.3286.

Precision The RSD % of cefmetazole by standard solution ranged from 0.7% to 1.9% and that of the polymer by sample ranged from 0.7% to 2.0% (Table I). The RSD % of repeatability and intermediate (inter-day) precision acceptance criteria for each QC was ≤2.0% [13]. The assay results successfully met the criteria.


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Table I. Precision of QC samples by HPSEC (n = 6)

Cefmetazole Polymer Repeatability Inter-day Repeatability Inter-day

Concentration(μg mL−1)

RSD %

Concentration(mg mL−1)

RSD % 1 1.9 1.8 0.25 1.4 2.0 10 0.7 1.7 1 0.7 1.4 50 0.9 1.0 1.5 1.2 1.3

Polymer Identification

The solution of cefmetazole sodium for injection (1 mg mL−1) was injected and analyzed by LC–MS. The polymer was eluted prior to cefmetazole through size-exclusion chromatography, and the strongest signal of the mass spectrometry ahead of cefmetazole was observed at m/z = 941 (Fig. 2a). After scanning the debris of the two grades, the following ion fragments

Fig. 2. Mass spectra of the polymer



201

were obtained: m/z = 115, 156, 322, and 354 (Fig. 2b). Based on the molecular weight of cefmetazole sodium, we speculated that m/z = 941 was produced by a dimer of two molecules of cefmetazole and m/z = 354 was the product after removing 1-methyl-1H-tetrazolium of -5-thiols (m/z = 115); meanwhile, the fragments of m/z = 322 and 156 were the products of the fragment of m/z = 354 (Fig. 3). These ion fragments further suggested that m/z = 941 was the polymer of cefmetazole.

Fig. 3. Proposed fragmentation pathways of the polymer

Stability

The amount of polymer at room temperature increased with extended time (Fig. 4), and the high-temperature test also indicates that temperature significantly influences the stability of cefmetazole (Fig. 1b). The formation of polymer is a dynamic process; thus, solutions for determination shall be detected immediately after preparation under low temperature. The results also suggest that cefmetazole sodium for injection should be allocated at low temperature in clinical practice and used immediately. Otherwise, the possibility of adverse reaction by polymer would increase.

Fig. 4. Peak area change of polymer along with time (mean ± SD, n = 6)


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Comparison of HPSEC and Gel Chromatography

Chromatographic conditions In consideration of the column used and polymer stability [7], phosphate buffer is recommended as the water mobile phase. Acetonitrile can be se-lected as the organic phase to improve the peak shape and ensure that the amount does not exceed 20% of the mobile phase, thereby protecting the column matrix. Four concentrations of phosphate buffer (0.0025, 0.005, 0.01, and 0.1 M) and four percentages of acetonitrile (5%, 10%, 15%, and 20%) in the mobile phase were investigated. Results showed that the highest sepa-ration effect was obtained when the phosphate buffer concentration was 0.01 M. Moreover, adding 10% acetonitrile further improved the separation effect. As in acidic and alkaline conditions, β-lactam antibiotics are prone to polymerization; thus, the pH of the mobile phase was set at 7.0. However, the chromatographic conditions for gel chromatography are relatively more complex than those for HPSEC because it needs mobile phase A (phosphate buffer) for testing the samples and mobile phase B (H2O) for testing the ref-erence standards on the Sephadex G-10 column, and the sample required more than 70 min of isocratic elution (Fig. 5), which is five times longer than that required for HPSEC.

Fig. 5. Gel chromatogram of the sample (1 — polymer, 2 — cefmetazole)



203

Chromatographic performances

The theoretical plate number and tailing factor of the polymer were 7853 and 0.97 for HPSEC. The theoretical plate number and tailing factor of blue dextran 2000 used to system suitability test for gel chromatography were 1209 and 1.81. The resolution value could not be compared because the cal-culation formulas between the two methods are different [12]. However, Figs. 1 and 5 obviously show that the baseline separation of HPSEC is better than that of gel chromatography. The results of chromatographic perform-ances indicate that HPSEC has better determination capacity for polymer amount than gel chromatography.

Linearity The equations of the linear regression line for cefmetazole and polymer on their respective working solutions and concentration range by gel chroma-tography were y = 0.4012x + 0.2131 and y =1.2157x + 0.3821, and the coeffi-cient of correlation was 0.9996 and 0.9988. The results indicate that the line-arity of cefmetazole and the polymer were better with HPSEC than with gel chromatography.

LOQ The LOQ of cefmetazole by gel chromatography was 25 ng mL−1. The HPSEC method could further develop a relatively low LOQ (1 ng mL−1), thereby making HPSEC potentially superior to gel chromatography.

Precision

The repeatability and intermediate (inter-day) precision of the two methods were compared by examining their respective QC samples. The results of the precision of gel chromatography are shown in Table II; the RSD % of cefmetazole by standard solution was less than 4.1% and that of the poly-mer by sample was less than 4.5%.

As shown in Tables I and II, the developed HPSEC method was more precise than gel chromatography.


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Table II. Precision of QC samples by gel chromatography (n = 6)

Cefmetazole Polymer

Repeatability Inter-day Repeatability Inter-day Concentration(μg mL−1)

RSD %

Concentration(mg mL−1)

RSD %

10 2.6 4.1 2.5 4.1 4.5 200 3.1 3.5 17.5 3.8 4.2 400 2.8 2.9 27.5 3.0 3.7

Application to determine the amount of polymer

Both methods were subsequently applied to assay seven batches of samples from Sichuan Hexin Pharmaceutical Co., Ltd. in China, and the results are shown in Table III. The mean amount of polymer from seven batches was 0.121% for HPSEC and 0.038% for gel chromatography. The admissibility limit for the polymer by gel chromatography is 0.1% [14], and the analytical results of the samples by gel chromatography met the criteria. The result of the Student t test indicated that the amount of polymer used in HPSEC was significantly higher than that in gel chromatography (p < 0.01), thereby suggesting the superior sensitivity of HPSEC and that the proposed method can be used to determine the criteria for limiting the amount of the polymer in cefmetazole.

Table III. Analytical results of samples by HPSEC and gel chromatography (n = 6)

Amount of polymer (mean %) Lot no.

HPSEC Gel chromatography 13080709 0.115* 0.034 13090703 0.073* 0.012 13090707 0.128* 0.039 13110703 0.141* 0.042 13110709 0.159* 0.076 13110723 0.083* 0.014 14060706 0.146* 0.051

Mean ± SD 0.121 ± 0.032* 0.038 ± 0.022

*p<0.01 (Student t test)



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Conclusions Few studies determined the amount of polymer in β-lactam antibiotic by using HPSEC. The method developed in this study can determine the amount of cefmetazole polymer with superior sensitivity and a shorter analysis time compared with gel chromatography. Thereby, the proposed HPSEC method can be used to measure the limited amount of polymer in cefmetazole, which could be recorded in pharmacopoeia, to control product quality.

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Development and Validation of a High-Performance Size