The investigation samples of Cefuroxime sodium were gifted by laboratory of APL Research Centre (A Division of Aurobindo Pharma Ltd, Hyderabad, India).

Chromatographic condition:

A HPLC system Waters alliance 2695 separation module equipped with a 2996 photodiode array detector along with Empower software for data acquisition and processing was used (Waters, Milford, USA), using stainless steel on YMC Pack ODS AQ column, 250mm x 4.6mm, 5µm particle size, (YMC, Inc.USA). The 0.02M phosphate buffer (by dissolving 2.72 g potassium dihydrogen orthophosphate in 1000ml of water adjusted to pH 2.8 ± 0.05 with diluted 20%v/v orthophosphoric acid), as mobile phase-A and LC grade methanol as Mobile phase-B. Initially 1.0ml min^-1 flow rate was set with injection volume of 20µl .The analysis was carried out under the gradient programme as follows, time (minutes)/A(v/v):B(v/v); T_0.01/100:0, T₁₀/100:0, T₁₅/20:80, T₂₅/20:80, T₂₆/100:0, T₄₀/100:0. This condition was established to elute all known related impurities of cefuroxime sodium, as these impurities elutes after 10 minutes of run time but no co-elution of impurities observed with lactic acid. Standard and test solution were prepared in Milli-Q water. The peak homogeneity was expressed in terms of peak purity values using Empower software, 2996 Photodiode array detector at 210nm.

Standard stock and sample solution:

Standard solution was prepared by dissolving accurately weighed 500mg of Lactic acid in 100ml. Diluted 5ml this solution in 100ml (250 µg ml^-1) as stock solution. Further, 4ml of this solution was diluted to 100ml (10 µg ml^-1) as standard solution, filtered through 0.45m or finer porosity membrane filter. Sample solution was prepared by dissolving accurately weighed 100mg of drug substance in 10ml, filtered through 0.45m or finer porosity membrane filter.

Chromatographic method development:

A simple potassium phosphate (KH₂PO₄) buffer with methanol as organic solvent at different concentrations were selected based on impurities elution, but in isocratic condition there was no separation of lactic acid with known related impurities of Cefuroxime sodium. The sample under investigation shows basic pH value based on literature. In view of that the buffer concentration was fixed by altering the solvent concentration, as cefuroxime sodium impurities were found to be sensitive towards organic modifiers the elution pattern also changes by variable concentration of solvents, based on the solvent polarity methanol was selected as organic phase. It is concluded that the methanol was not allowed into the chromatograph up to 10 minutes interval of interval, the impurities which are sensitive to methanol are un-retained by column stationary phase. When the concentration of methanol varied to 80% level at 15 minutes interval, the related other impurities were eluted, the acquisition time also extended based on peak retention. The method is optimized in such a way that the separation and base line were good with no co-elution of impurities with lactic acid.

In the described experiment, the effect of pH and organic modifier on retention and elution were studied. When the pH of buffer was varies from 3.0 and 2.6 with diluted ortho phosphoric acid, a 0.5-1.0 minute variation in retention on either side with slight co-elution of cefuroxime sodium impurities. It was concluded that the buffer solution pH of 2.8 ± 0.05 to be fixed with no initial organic solvent composition was found to be ideal. The initial data acquisition up to 10 minutes of chromatogram is considered for lactic acid determination, the gradient time from 11 minutes to 40 minutes taken as injection delay, because in this investigation the peak of interest is lactic acid determination. But in the case of specificity analysis total 40 minutes run time is considered to check the retention time for all known related impurities of Cefuroxime sodium. The effect of column oven temperature also studied at variable points. Slight decreases in resolution of lactic acid with known impurities were noticed towards higher side. Hence, it is concluded that the best result were achieved when the temperature was ambient. From overall chromatographic conditions determination of lactic acid was established by using 0.02M KH₂PO₄ and methanol with gradient chromatographic programme respectively, with column oven temperature of 25°C to optimized the method. The Figure 2(a) represents a typical chromatogram of lactic acid standard. The proposed HPLC method was validated for specificity, linearity, accuracy, limit of detection and limit of quantification, intermediate precision, stability of sample solution, stability studies and forced degradation studies

RESULTS AND DISCUSSION:

Validation of the method:

Selectivity:

The sample solutions of impurities, sample and standard were prepared at 1.0%w/w concentration based on Cefuroxime sodium and injected into the chromatographic system to identify the retention time. The retention time of Lactic acid was found to be about 5.35 min. However, due to high concentration of Cefuroxime, it does not elute at the acquisition time, so the known related impurities of cefuroxime were eluting in the gradient programme. The sample was found to contain Lactic acid at very low level, and therefore, the sample (Cefuroxime sodium) was spiked with Lactic acid at 0.3% w/w level along with other known impurities of Cefuroxime sodium, other known impurities, thereby indicating that the method is selective for determining the content of Lactic acid. In view, overlay chromatogram of diluent, standard, spiked sample and sample spiked with all known related impurities including Lactic acid is enclosed as Figure 2(b).

Sensitivity:

The solutions were prepared from known stock concentration to predict the limit of detection (LOD) and limit of quantification (LOQ).The values were predicted using slope (S) and residual standard deviation (S.D) obtained from a linear regression line performed at lower concentration levels. The predicted limit of detection and quantification was found to be 3.3 µg ml^-1and 10.1 µg ml^-1 respectively, and each predicted level was verified for precision by analyzing six replicate measurements. The percentage relative standard deviation for six replicate measurements at predicted LOD and LOQ concentration levels was found to be 6.6 and 13.0 respectively, verifying the predicted values.

Linearity:

Linearity was validated by measuring area responses at different levels of lactic acid over the range of 20% to 150% of analyte concentration. The solutions were prepared from stock solutions of required concentration for six different levels of 0.6, 1.5, 2.3, 3 0, 3.8 and 4.5µg ml^-1, correlation co-efficient was greater than 0.9984. The %RSD values of the slope and Y-intercept of calibration curve was calculated. The area and concentration treated were treated by least squares linear regression analysis. The linearity data of statistical analysis is tabulated in Table 1.

Table 1: Statistical evaluation of Linearity

^a CC: Correlation coefficient

Component	Lactic acid
Calibration range (µg ml ^-1)	6.078 - 45.586
Calibration Points	6
Slope	584
STEY X	552
Intercept	2926
CC¹(r)	0.9984

Precision:

The system precision for the method was assessed by six replicate injections of Lactic acid standard solution (10 mg ml^-1) into chromatographic system, and the percentage relative standard deviation of response for six replicate measurements was found to be 1.1.Repeatability of the method (Method precision) was demonstrated by preparing six replicate sample preparations by spiking known concentration (0.3% w/w) of Lactic acid in random selection of one lot Cefuroxime sodium drug substance.

Table 2: Experimental data of precision and system suitability

Component	System suitability	Lactic Acid Amount (%w/w)^d	%R.S.D
Retention Time (R_T) ^b	5.8	-	-
Asymmetry Factor^b	1.1	-	-
Theoretical Plates^b	11500	-	-
System Precision^c	-	-	1.1
Method Precision^c	-	0.35	0.8
Intermediate Precision^c	-	0.34	1.5

^b Average data of overall experimental observation, ^cn=6, average of six determinations, ^d Average

These were analyzed as per method, and the content of Lactic acid was determined. Intermediate precision of the method (Ruggedness) was performed in the same way as described in method precision, however, by employing different analyst on other day using another lot of chromatograph. The content of Lactic acid was determined in each preparation, and the percentage relative standard deviation for six replicate measurements was found to be 1.5. The experimental results are summarized with system suitability parameters were presented in Table-2.

Stability of sample solution:

The sample solution prepared by spiking known concentration (0.3% w/w) of Lactic acid with respect to sample concentration was stored at 25 ± 2°C temperature conditions, and was injected into chromatographic system at different time intervals. The content of Lactic acid was determined at each interval, the sample solution was found to be stable over a period of 12 hours. The % difference between the peak area obtained at initial and different time interval was found to be less than 1.0. However, the sample solution was found to be stable for at least 12 hour at room temperature (~25°C).

Accuracy:

The accuracy of the method was evaluated by preparing sample solution spiked with known amount of Lactic acid at different concentration levels in the range between 20%, 50%, 100% and 150% with respect to Cefuroxime concentration. Each concentration of sample solution was prepared in triplicate and analyzed as per the method. The percent recovery of Lactic acid was found to be in the range of 98.1 to 101.9, mean percent recovery was 99.4, when calculated against the known added amount, indicating that the method is accurate. The results of accuracy experiment are tabulated in Table 3.

Robustness

To assess the robustness of the method, experimental conditions were deliberately altered. The experimental condition altered by changing the buffer pH between (2.6-3.0), temperature (20°C-30°C), flow of mobile phase (0.9-1.0 mL min^-1) and wavelength (205-215nm). The result obtained from the robustness indicated that, the experimental method parameters were tolerance limit with minor changes to optimize the method.

Stability Studies

To present stability studies on Cefuroxime sodium drug substance for the determination of lactic acid content, the analysis were conducted on samples from variable sources of temperature and humidity storage of accelerated (40°C/75%RH), long term (25°C/60%RH) and refrigerated (2-8°C) storage condition¹². The results obtained from accelerated storage condition of 6 months samples were shown to be 0.11%w/w, 0.13%w/w and 0.09%w/w respectively. Where as in the case of sample stored at 36 months station of long-term period also shows 0.09%w/w, 0.12%w/w and 0.11%w/w comparatively similar result were obtained for refrigerated samples. Hence formation of lactic acid in cefuroxime sodium drug substances resulting as process related impurity, in view of that the sample shows no degradation profile with respect to storage at different conditions of temperature and humidity, The experimental condition shows precise results with good repeatability on different days with other analyst, shows the method is rugged for the determination of Lactic acid.

Forced degradation studies

The degradation study conducted on different condition of light, thermal and humidity for dry exposure. Liquid phase degradation using acid, base and peroxide of different concentration also conducted to prove the method is stability indicating shows no degradation on lactic acid in cefuroxime sodium drug substance.

The UV light exposed up to 200 watts/sq.mtr, fluorescent light exposed up to 1.2 million lux hour, Humidity exposure at 25°C / 92%RH, thermal exposure at 105°C and 60°C with the time duration of 72 hours and hydrolysis using aqueous media up to 10 hour at room temperature were performed to these condition. The result obtained from the chromatographic data shows no degradation for lactic acid and found to be below limit of detection (below 0.033%w/w). In addition, wet stress condition also conducted using 30% H₂O₂(after 2 hours),5M Hydrochloric acid (after 2 hours), 5M sodium hyroxide (after 2 hours) were added separately for sample taken into different reservoir were studied, the observation value shows no degradation of Lactic acid and the values were found to be 0.009% to 0.016%w/w (below limit of detection). Hence, the method is found to be selective and stability indicating with respect to forced degradation data.

CONCLUSION:

The proposed HPLC method developed for quantitative determination of lactic acid in Cefuroxime sodium drug substance is rapid, accurate, precise and selective .the method was showing satisfactory validation data for the tested parameters as per the ICH guidelines. The proposed method is simple and cost effective as it uses commonly used ODS column under gradient elution, with moderate run time. Hence the proposed method is conveniently used by quality control department for the determination of lactic acid content in Cefuroxime sodium drug substance.

ACKNOWLEDGEMENTS:

The authors express their sincere thanks to APL Research Centre [A division of Aurobindo Pharma Ltd.] located at Hyderabad, India for providing the analytical support to pursue this work.

REFERENCES:

1. International Conference on Harmonization of Technical Requirement for Registration of Pharmaceuticals for Human use. ICH harmonized tripartite guideline. Validation of Analytical procedures Text and methodology Q2 (R1), 2005.

2. The United States Pharmacopeial conventions, 1601-Twin brook rockways, Rockville, MD 2085. USP monograph: Cefuroxime Sodium, USP 32-NF 27; 2009; 1865-1867.

3. Cefuroxime sodium Ph.Eur monograph 0992, British Pharmacopoeia, Volume I; 2009; 411-412.

4. International conference on harmonization Q3A (R2): Draft revised Guidance on impurities in new drug substances; 2006.

5. The United States Pharmacopeial conventions, Lactic acid; USP 32-NF 27; 2009; 2745-2746.

6. Ph.Eur.monograph:0458, British Pharmacopeia, BP-2009, Volume I, 1181-1182.

7. Li Tan, Yu Wang et. al, Simultaneous determination of L-and D-lactic acid in plasma by capillary electrophoresis, Journal of Chromatography B, 2005; 814; 393-398.

8. John Savory, Alex Kaplan, A gas chromatographic method for the determination of lactic acid in blood, Clinical Chemistry,1966; 12; 559-569.

9. R.J.Buswell, The determination of lactic acid in shortening containing lactylated glycerides, by liquid –liquid partition chromatography, Journal of the American oil chemist society, 1964; 41; 457-459.

10. John McHugh, et al; New and simple HPLC method for the determination of Lactic acid content in Ciprofloxacin Injection, Journal of Liquid Chromatography & Related Technologies, June 2006; 29, 1905-1916.

11. L.R.Snyder, J.J.Krikland and J.L.Glajch. Practical HPLC Method Development; IIed, John Wiley & Sons, New York, NY, 1997, 236-260.

12. International Conference on Harmonization of Technical Requirement for Registration of Pharmaceuticals for Human use. ICH harmonized tripartite guideline, Stability testing of new drug substances and products Q1A (R2) February 2003.

Component	LACTIC ACID
	Amount (% w/w)
Specification level^e	Added ^f	Found ^f	% Recovery ^g	%R.S.D^h
20% of specification level	0.063	0.062	98.4	0.9
50% of specification level	0.149	0.152	101.9	0.4
100% of specification level	0.302	0.298	98.7	1.0
150% of specification level	0.453	0.444	98.1	1.2

Overall statistical data	Mean		99.4
	SD		1.63
	% R.S.D		1.6
	95% Confidence level (±)		± 2

INTRODUCTION: