A Simple and Modified Method Development of Vancomycin Using High
Journal of Chromatography & Separation Techniques

Journal of Chromatography & Separation Techniques
Open Access

ISSN: 2157-7064

+44 1300 500008

Research Article - (2015) Volume 6, Issue 7

A Simple and Modified Method Development of Vancomycin Using High Performance Liquid Chromatography

Meenakshi K Chauhan* and Nidhi Bhatt
NDDS Research Laboratory, Department of Pharmaceutics, Delhi Institute of Pharmaceutical Sciences and Research, New Delhi, India
*Corresponding Author: Meenakshi K Chauhan, NDDS Research Laboratory, Department of Pharmaceutics, Delhi Institute of Pharmaceutical Sciences and Research, Government Of NCT Of Delhi, Pushp Vihar, Sec-3, New Delhi-110 017, India Email:


Vancomycin is well known as a prominent member of the glycopeptide class of antibiotics. In this paper, a rapid resolution method using high performance liquid chromatography is employed to identify the presence of Vancomycin. A robust method is established and validated for the simultaneous quantification of glycopeptides antibiotic like Vancomycin and applied for their pharmaceutical research. This method is simple, modified, user and environment friendly with new techniques and all sample preparations are performed in excellent manner. The method showed good sensitivity with 2 μg/mL limit of quantification (LOQ) and the calibration curve is linear in the range of 2-500 μg/mL concentration range. The within- run and between- run precision obtained is less than 2 and 4% respectively. The method is proposed to be applied in an experimental micro particulate carrier based oral study design. The results can proved to be specific, linear, accurate and sensitive and the available data can provide valuable information regarding the analysis and pharmacokinetics of Vancomycin after the oral administration.

Keywords: Vancomycin; Glycopeptide; High performance liquid chromatography; Antibiotics


Vancomycin (VCM) is a glycopeptide antibiotic which is widely employed in the treatment of serious infections by Gram positive bacteria [1,2]. It was chosen for therapy of infections in patients allergic to β-lactam antibiotics. This antibiotic acts by preventing the peptidoglycan synthesis of bacterial cell wall [3]. Monitoring VCM in the biological fluids and in pharmaceutical products is of importance to prevent side effects in patients under treatment and to achieve optimum therapeutic concentrations [4]. Moreover, antibiotics including VCM have been found in the aquatic environment such as waste and polluted water resources. This compound can play a role in the maintenance or extension of antibiotic resistance bacteria, finally resulting in hazards to human health [5,6]. Therefore, screening VCM both in biological and environmental studies seems to be very important. Several different approaches in liquid chromatography have been developed to satisfy the demand for fast analysis without compromising separation efficiency and resolution. The main efforts have been focused on performing separations at high performance liquid chromatography [7].

The aim of our study is to develop a modified HPLC method for measuring total VCM concentrations with acceptable runtimes. The current method is easier to carry out the qualitative and quantitative analysis of vancomycin in vivo, by using new improved mobile phases in very short time. In addition, the clinical impact of developed method is planned to be investigated with the new oral formulation in our lab for validating the outcomes. This study might play a very significant role in certain therapeutic effects and warrant further study in the case of oral micro particulate based oral study design.


Chemicals and materials

VCM was provided by Concord Biotech Limited. HPLCgrade acetonitrile and Formic acid were obtained from RFCL limited. Water was purified by a Milli-Q system (Millipore, Billerica, MA, USA). All the used chemical reagents were of analytical grade and were used as received.

Instruments and chromatographic conditions

Chromatographic experiments were performed on Shimadzu UFLC system (SPD-M20A) equipped with a binary pump (model LC- 20AD) with a diode array detector (Gro-Zimmern, Germany). The separation was carried out on Nucleosil 120 C18 5 μm column at 35°C with a flow rate of 1 mL/min and Injection volume was 25 μL. Mobile phase was a mixture of Acetonitrile (A): Water, pH 2.0 (B) and Formic acid was used to adjust the pH of water. For data processing, a Class VP Data system (Shimadzu, Duisburg, Germany) was used. Binary pump was used for carrying two different solvents.

Preparation of standards, calibration standard and test samples

Standard stock solutions were prepared in ultra pure water. The stock solution was further diluted with water in formation of the following standard solutions, with the concentration of 2-20, 40-300 and 10-500 μg/mL, respectively. All the freshly prepared solutions were used for analysis. The chromatograms of different standard solutions are shown in Figure 1. The chromatograms were identified in linear relationship by their retention times and a response height with respect to concentration for the different calibration ranges which are put up in Figure 2.


Figure 1: Chromatogram of VCM standard solution.


Figure 2: The response height versus concentration curve of VCM.

Results and Discussion

Optimization of chromatographic condition

To obtain reliable chromatographic results and appropriate ionization, several mobile phase systems (acetonitrile-0.2 M sodium sulfate buffer, acetonitrile-10 mM acetate buffer, pH 4.0, acetonitrilewater etc.) were tested and compared [8-19]. The results suggested that acetonitrile-acid aqueous solution was superior to the others. Meanwhile, formic acid was added into the mobile phase for pH adjustment and to improve the peak shape as well as to restrain the peak tailing. This finding was confirmed by the optimal solvent systems containing a mixture of 0.1% formic acid-acetonitrile (A) and water (B). For obtaining the effective separation, gradient elution technique was employed which guarantees high ionization, and minimize the ion suppression.

Optimization of sample preparation

Different methods of sample preparation were tested to select a coherent extraction method for VCM. Different volume ratios (50:50, 80:20, 70:30) of mobile phases were investigated. The results suggested that the volume ratio 50:50 was best among others.

Screening and analysis of optimized samples

The optimized samples were screened and analyzed. The results concluded that the retention time (2.5 min) for the drug was very less as compared to other previously reported methods in literature. The drug was very well separated and identified by their less retention time, with remarkable peak heights.

Method validation

The method was validated with regard to its specificity, linearity, accuracy and sensitivity (within and between days) [20-25].

Linearity and LOQ: Regression equations, linear ranges, correlation of coefficients are shown in Table 1. All calibrations consists excellent linearity with coefficients (r) higher than 0.995. As seen in Table 1 LOQ of three ranges were 2, 40 and 10 μg/mL respectively [26], with accuracy (recovery) between 74.5% and 111% this was absolute for quantification study. The correlation coefficient calculation and the regression analysis were performed without any type of mathematical transformation [27-31].

Linear range (µg/mL) Slope Intercept Correlation coefficient (r)
2-20 18612 22180 0.996
40-300 1403 8350 0.997
10-500 2036 10654 0.996

Table 1: Regression data and statistical analysis.

Extraction recovery: For checking the accuracy of the method, three standard samples with low intermediate and high concentrations of each range were analyzed. The concentrations were calculated from the corresponding calibration standard line (experimental concentrations) and were compared with the theoretical concentrations [32-38]. The extraction recovery was estimated according to Equation 1 and is shown in Table 2.

Measure conc. (µg/mL) Accuracy(% Recovery)
2 74.5
10 95.5
20 101
40 75
150 105
300 102
10 111
250 92.4
500 83

Table 2: Recovery Data.

Recovery=((Cexp/Cteo) × 100) ....1

Where Cexp=Experimental concentration and

Cteo=Theoretical concentration

Assessment of precision, repeatability and reproducibility are listed in Tables 3 and 4, which were calculated on the same day and on 6 different days. Intra and inter- precision (relative standard deviation, RSD) of samples was less than 1.72% and 3.12%, respectively.

Spiked conc. (µg/mL) Mean response ± SD Intra-run (RSD%)
2 57164.4 ± 793.907 1.38
4 74595.83± 1288.45 1.72
10 221093.3 ± 3629.48 1.64
100 158255.2 ± 1668.99 1.05
200 296663 ± 2399.00 0.80
300 428491 ± 1244.61 0.29
400 887793.1 ± 7134.58 0.80
500 1122905 ± 8527.87 0.75

Table 3: Repeatability (Intra-run precision). SD: Standard deviation; RSD: Relative standard deviation.

Spiked conc. (µg/mL) Mean response ± SD Inter-run (RSD%)
2 57333.27 ± 1109.148 1.93
4 75966.8 ± 2371.276 3.12
10 221859.6 ± 5534.238 2.49
100 160598.5 ± 4399.284 2.73
200 293177 ± 2511.768 0.85
300 428532.9 ± 2820.084 0.65
400 892793.1 ± 7691.935 0.86
500 1119745 ± 8069.782 0.72

Table 4: Reproducibility (Inter-run precision).SD: Standard deviation; RSD: Relative standard deviation.


The chromatographic system was proved to be a rapid and efficient for research purpose of VCM formulation in systemic use. The method was established and validated for the simultaneous quantification of the drug and can be applied for their pharmacokinetic research. These works could provide more in- depth knowledge into the active components working in vivo with VCM and would be helpful for further investigation regarding the pharmacology and mechanism of VCM. This study would be helpful for explaining the metabolism of drug in rat and human plasma.


  1. Nicolaou KC, Boddy CN, Bräse S, Winssinger N (1999) Chemistry, Biology, and Medicine of the Glycopeptide Antibiotics. AngewChemInt Ed Engl 38: 2096-2152.
  2. Nailor MD, Sobel JD (2011) Antibiotics for gram-positive bacterial infection: vancomycin, teicoplanin, quinupristin/dalfopristin, oxazolidinones, daptomycin, telavancin and ceftaroline. Med Clin North Am 95: 723-742.
  3. Allen NE, Nicas T (2003) Mechanism of action of oritavancin and related glycopeptide antibiotics. FEMS Microbiol Rev 26: 511-532.
  4. Zhang T, Watson DG, Azike C, Tettey JN, Stearns AT, et al. (2007) Determination of vancomycin in serum by liquid chromatography-high resolution full scan mass spectrometry. J Chromatogr B AnalytTechnol Biomed Life Sci 857: 352-356.
  5. Li B, Zhang T, Xu Z, Fang HH (2009) Rapid analysis of 21 antibiotics of multiple classes in municipal wastewater using ultra performance liquid chromatography-tandem mass spectrometry. Anal ChimActa 645: 64-72.
  6. Tamtam F, Mercier F, Le Bot B, Eurin J, TucDinh Q, et al. (2008) Occurrence and fate of antibiotics in the Seine River in various hydrological conditions. Sci Total Environ 393: 84-95.
  7. Belal F, el-Ashry SM, el-Kerdawy MM, el-Wasseef DR (2001) Voltametric determination of vancomycin in dosage forms through treatment with nitrous acid. Arzneimittelforschung 51: 763-768.
  8. Jesús Valle MJ, López FG, Navarro AS (2008) Development and validation of an HPLC method for vancomycin and its application to a pharmacokinetic study. J Pharm Biomed Anal 48: 835-839.
  9. Bijleveld Y, de Haan T, Toersche J, Jorjani S, van der Lee J, et al. (2014) A simple quantitative method analysingamikacin, gentamicin, and vancomycin levels in human newborn plasma using ion-pair liquid chromatography/tandem mass spectrometry and its applicability to a clinical study. J Chromatogr B AnalytTechnol Biomed Life Sci 951-952: 110-118.
  10. Júnior AR, Vila MMDC, Tubino M(2008) Green spectrophotometric method for the quantitative analysis of vancomycin in pharmaceuticals and comparison with HPLC. Anal Lett 41:822-836.
  11. El-Ashry SM, Belal F, El-Kerdawy MM, El Wasseef DR(2000) Spectrophotometric Determination of Some Phenolic Antibiotics in Dosage Forms. MicrochimActa 135: 191-196.
  12. Vila MMDC, Salomão AA, Tubino M, (2008)Eclectic Chemistry. Eclet Quim 33: 67-72.
  13. El-Didamony AM, Amin AS, Ghoneim AK, TelebanyAM(2006) Indirect spectrophotometric determination of gentamicin and vancomycin antibiotics based on their oxidation by potassium permanganate. Cent Eur J chem 4: 708-722.
  14. Sastry CSP, Rao TS, Rao PSNHR, Prasad UV(2002) Assay of Vancomycin and Dobutamine Using Sodium Metaperiodate. MicrochimActa 140: 109-118.
  15. Pfaller MA, Krogstad DJ, Granich GG, Murray PR (1984) Laboratory evaluation of five assay methods for vancomycin: bioassay, high-pressure liquid chromatography, fluorescence polarization immunoassay, radioimmunoassay, and fluorescence immunoassay. J ClinMicrobiol 20: 311-316.
  16. Lam MT, Le Chris X (2002) Competitive immunoassay for vancomycin using capillary electrophoresis with laser-induced fluorescence detection. Analyst 127: 1633-1637.
  17. Tetin SY, Swift KM, Matayoshi ED (2002) Measuring antibody affinity and performing immunoassay at the single molecule level. Anal Biochem 307: 84-91.
  18. Furuta I, Kitahashi T, Kuroda T, Nishio H, Oka C, et al. (2000) Rapid serum vancomycin assay by high-performance liquid chromatography using a semipermeable surface packing material column. ClinChimActa 301: 31-39.
  19. López KJ, Bertoluci DF, Vicente KM, Dell'Aquilla AM, Santos SR (2007) Simultaneous determination of cefepime, vancomycin and imipenem in human plasma of burn patients by high-performance liquid chromatography. J Chromatogr B AnalytTechnol Biomed Life Sci 860: 241-245.
  20. Hagihara M, Sutherland C, Nicolau DP (2013) Development of HPLC methods for the determination of vancomycin in human plasma, mouse serum and bronchoalveolar lavage fluid. J ChromatogrSci 51: 201-207.
  21. Backes DW, Aboleneen HI, Simpson JA (1998) Quantitation of vancomycin and its crystalline degradation product (CDP-1) in human serum by high performance liquid chromatography. J Pharm Biomed Anal 16: 1281-1287.
  22. Farin D, Piva GA, Gozlan I, Kitzes-Cohen R (1998) A modified HPLC method for the determination of vancomycin in plasma and tissues and comparison to FPIA (TDX). J Pharm Biomed Anal 18: 367-372.
  23. Saito M, Santa T, Tsunoda M, Hamamoto H, Usui N (2004) An automated analyzer for vancomycin in plasma samples by column-switching high-performance liquid chromatography with UV detection. Biomed Chromatogr 18: 735-738.
  24. Ye G, Cai X, Wang B, Zhou Z, Yu X, et al. (2008) Simultaneous determination of vancomycin and ceftazidime in cerebrospinal fluid in craniotomy patients by high-performance liquid chromatography. J Pharm Biomed Anal 48: 860-865.
  25. Tariq A, Siddiqui MR, Kumar J, Reddy D, Negi PS, et al. (2010) Development and validation of high performance liquid chromatographic method for the simultaneous determination of ceftriaxone and vancomycin in pharmaceutical formulations and biological samples. Sci Asia 36: 297-304.
  26. Favetta P, Guitto J, Bleyzac N, Dufresne C, Bureau J (2001) New sensitive assay of vancomycin in human plasma using high-performance liquid chromatography and electrochemical detection. J Chromatogr B Biomed SciAppl 751: 377-382.
  27. Shibata N, Ishida M, Prasad YV, Gao W, Yoshikawa Y, et al. (2003) Highly sensitive quantification of vancomycin in plasma samples using liquid chromatography-tandem mass spectrometry and oral bioavailability in rats. J Chromatogr B AnalytTechnol Biomed Life Sci 789: 211-218.
  28. Cheng C, Liu S, Xiao D, Hollembaek J, Yao L, et al. (2010) LC-MS/MS method development and validation for the determination of polymyxins and vancomycin in rat plasma. J Chromatogr B 878: 2831-2838.
  29. Seifrtová M, Nováková L, Lino C, Pena A, Solich P, et al. (2009) An overview of analytical methodologies for the determination of antibiotics in environmental waters. Anal ChimActa 649: 158-179.
  30. Abu-Shandi KH (2009) Determination of vancomycin in human plasma using high-performance liquid chromatography with fluorescence detection. Anal BioanalChem 395: 527-532.
  31. Musenga A, Mandrioli R, Zecchi V, Luppi B, Fanali S, et al. (2006) Capillary electrophoretic analysis of the antibiotic vancomycin in innovative microparticles and in commercial formulations. J Pharm Biomed Anal 42: 32-38.
  32. Bonnici PJ, Damen M, Waterval JC, Heck AJ (2001) Formation and efficacy of vancomycin group glycopeptide antibiotic stereoisomers studied by capillary electrophoresis and bioaffinity mass spectrometry. Anal Biochem 290: 292-301.
  33. LeTourneau DL, Allen NE (1997) Use of capillary electrophoresis to measure dimerization of glycopeptide antibiotics. Anal Biochem 246: 62-66.
  34. Heinisch S, Rocca JL (2009) Sense and nonsense of high-temperature liquid chromatography. J Chromatogr A 1216: 642-658.
  35. Yea G (2008) Simultaneous determination of vancomycin and ceftazidime in cerebrospinal fluid in craniotomy patients by high-performance liquid chromatography. J Pharm Bio Analysis 48: 860-865.
  36. Kitahashi T, Furuta I (2001) Determination of vancomycin in human serum by micellarelectrokinetic capillary chromatography with direct sample injection. ClinChimActa 312: 221-225.
  37. Hefnawy MM, Sultan MA, Al-Shehri MM (2007) HPLC separation technique for analysis of bufuralol enantiomers in plasma and pharmaceutical formulations using a vancomycin chiral stationary phase and UV detection. J Chromatogr B AnalytTechnol Biomed Life Sci 856: 328-336.
  38. Diana J, Visky D, Roets E, Hoogmartens J (2003) Development and validation of an improved method for the analysis of vancomycin by liquid chromatography selectivity of reversed-phase columns towards vancomycin components. J Chromatogr A 996: 115-131.
Citation: Chauhan MK, Bhatt N (2015) A Simple and Modified Method Development of Vancomycin Using High Performance Liquid Chromatography. J Chromatogr Sep Tech 6:296.

Copyright: © 2015 Chauhan MK, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.