Original Article Performance Verification Test of High Performance Liquid Chromatography: Practical Example
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1 Available online at International Journal of Chromatographic Science Universal Research Publications. All rights reserved Original Article Performance Verification Test of High Performance Liquid Chromatography: Practical Example Lantider Kassaye, Getachew Genete Food, Medicine and Health Care Administration and Control Authority, Food and Drug quality Control Laboratory, Addis Ababa, Ethiopia Received 18 January 2013; accepted 11 March 2013 Abstract The performance of an HPLC system can be evaluated by examining the key functions of the various modules that comprise the system. The pump, auto injector, column oven and the detector are the most important parts of an HPLC system which needs to be verified for the proper functioning. Flow rate accuracy and gradient accuracy for the pump; Precision, Linearity and carry over for the auto sampler; wavelength accuracy and response linearity for the detector and temperature accuracy for the column oven are the most important parameters which need to be considered during HPLC performance verification. In this practical example, all of the modules except column oven (for more than 50 o C) meet the acceptance/refusal criteria suggested by Herman Lam Universal Research Publications. All rights reserved Key words: Verification of HPLC; HPLC; Performance qualification of HPLC Introduction Good analytical results are essential in order to take reliable decisions. Analytical measurements affect the daily lives of every citizen. Sound, accurate and reliable analytical measurements are fundamental to the functioning of modern society. A wrong result can have an enormous social and economic impact [1]. The correctness of measurements and measuring instruments is one of the key prerequisites to ensure the quality of products and services, and the accuracy of the instruments must be consistent with their intended use [2]. Calibration and verification are the most important actions to ensure the correct indication of measuring instruments [2]. Regular calibration of measuring instruments should be carried out in agreement with the implemented quality systems. The industrial metrology ensures the appropriate functioning of measurement instruments used in industry as well in production and testing processes, in order to guarantee the quality of life for citizens and for academic research [3]. Verification is the confirmation, based on evidences (facts, test results) that some specified requirement has been fulfilled. The result from a verification assay will show if the measuring equipment is in agreement with its required specifications, which are generally expressed as tolerances [4]. The verification of measuring instruments includes testing and requires the availability of clear specifications and acceptance/refusal criteria [5]. Verification provides means of checking that the deviations between the values displayed by a measuring instrument and the corresponding known values of measured quantity are under control. High-Performance Liquid Chromatography (HPLC) is one of the foremost analytical techniques widely used in analytical laboratories for the analysis of pharmaceuticals and chemicals [6-8], foods [9-11], cosmetics samples and so on [12, 13]. In order to provide a high level of assurance that the data generated from the HPLC analysis are reliable, the performance of the HPLC system should be monitored at regular intervals. The performance of an HPLC system can be evaluated by examining the key functions of the various modules that comprise the system. The common HPLC performance attributes and the acceptance/refusal criteria are presented in Table 1. The acceptance/refusal criteria values for these attributes are based on the values suggested by Herman Lam in the chapter Performance Verification of the HPLC of his book [14]. 2. Experimental 2.1 Instruments and apparatuses A low pressure quaternary HPLC instrument comprises LC-10ATvp pump, SIL-10ADvp auto sampler, SPD- 10AVvp UV-Vis detector and CTO-10AVP column oven (Shimadzu, Japan) has been verified for its proper functioning. Analytical balance (METTLER Toledo, Switzerland), Calibrated stop watch (Treaceble stopwatch, JUMBO DIGIT, VWR, China) and thermometer (Service Testo, Germany) have been used. 2.2 Materials and chemicals ODS analytical column (150 X 4.6 mm, 5μm, Waters, USA), Class A 25mL volumetric flasks (Pyrex, Germany), 18
2 methanol HPLC grade (BDH, USA), Acetonitrile HPLC grade (J.T. Baker, USA) and caffeine (Fluka, Sigma- Aldrich, China, purity 0.99%) were employed for this work. Table 1: Performance attributes and their acceptance/ refusal criteria for HPLC verification Module Performance Acceptance/refusal Attributes criteria Pump Flow rate accuracy ± 1% of the set flow rate Gradient accuracy ± 1% of the step gradient composition Auto injector Precision RSD 1% Linearity r Carryover < 1% Detector Wave length accuracy ± 2nm Linearity of r Column oven response Temperature accuracy ± 2 o C of the set temperature 2.3 Methods Chromatographic condition (For Auto sampler and Detector verification) A mobile phase containing 85% water and 15% acetonitrile was pumped through the analytical column (ODS, 150 X 4.6mm, 5μm) at a flow rate of 2 ml/minute. The test solutions, caffeine in methanol, were injected and detected at 272nm. The column temperature was ambient Verification Test for different modules of HPLC Pump Module: Flow Rate Accuracy The flow-rate of the pump was set at 2 ml/min. and the time required to fill a 25 ml volumetric flask was measured using the calibrated stopwatch. Gradient Accuracy and Linearity The accuracy and linearity of the gradient solvent delivery has been verified indirectly by monitoring the absorbance change as the binary composition of the two solvents changes from two different channels. The LC gradient has four channels: A, B, C and D. The test was performed for two channels at a time. Channel A is filled with a pure solvent, methanol, while channel B is filled with a solvent containing a UV-active tracer, caffeine. The gradient profile is programmed to pump from solvent A only for two minutes and then to decrease the composition of Solvent A by 10% and to increase solvent B by the same percent and allowing to pump for two minutes at each change. The absorbance change at each composition change is measured and expressed as height H in the plot of absorbance versus solvent composition. The linearity of the gradient delivery was verified by plotting the absorbance at various mobile phase compositions versus the theoretical composition. The entire process was repeated for channels C and D Injector Module: Precision The precision of the injector was demonstrated by making six replicate injections from a sample (caffeine 0.05 mg/ml in methanol). The relative standard deviation (RSD) of the response of the injections was calculated to evaluate the precision. Linearity The linearity of the injector was demonstrated by making injections of 5, 10, 20, 30 and 50 μl of the caffeine solution (0.05 mg/ml in methanol). The response of the injections at each injection volume was plotted against the injection volume. The correlation coefficient of the plot was used in the evaluation of the injection linearity. Carryover Carryover was evaluated by injecting a blank (methanol) after a sample that contains a high concentration of analyte ((0.2 mg/ml of caffeine in methanol). The response of the analyte found in the blank sample expressed as a percentage of the response of the concentrated sample was used to determine the level of carryover UV-Visible Detector Module: Wavelength Accuracy The wavelength verification of the UV range was performed by filling a flow cell with a Caffeine solution (0.015mg/mL in methanol) and the UV spectrum was collected in the range of 200 to 400nm. The obtained scan should have λ max at about 272nm and λ min at about 244nm. Linearity of Response The linearity of the detector response was checked by injecting a series of standard solutions of caffeine (0.05, 0.1, 0.2, 0.4 and 1 mg/ml) to the chromatographic system. From the plot of response versus the concentration of the solutions, the correlation coefficient between sample concentration and response was calculated to determine the linearity Column Oven module: The accuracy of the oven was checked at 30, 40, 50, 60 and 70 o C using the calibrated thermometer. The correction factors obtained from the calibration certificate of the thermometer were considered to get the experimental temperature. Results and Discussion 1. Pump module 1.1. Flow rate Accuracy One of the key performance requirements for the pump module is the ability to maintain accurate and consistent flow of the mobile phase, which will be necessary to provide stable and repeatable interactions between the analytes and the stationary phase [15]. Poor flow-rate accuracy will affect the retention time of the separation. As shown in table 2, the results for flow rate accuracy for all of the four suction channels is in the range of to % and this good result indicates that the pump performance is superior Gradient Accuracy and Linearity For gradient accuracy, the ability of the pump to deliver the mobile phase at different solvent strengths over the time by varying the composition of the mobile phase accurately is fundamental to get the adequate chromatographic separation and reproducibility. As shown in fig 1, the absorbance increased step by step as the composition of the caffeine increased. Similarly, the 19
3 absorbance decreased to zero as the composition of the caffeine decreased to zero. The height increased due to the change in caffeine composition was measured by the LC solution software from the baseline and these heights have been used for the determination of gradient accuracy and gradient linearity. The gradient linearity is determined by graphing the percentage composition of caffeine versus the respective obtained height. As shown in figure 2, the relationship between percentage of the composition of Table 2: Flow rate accuracy test result the four pump channels separately. a directly measured by the stopwatch b converted to minutes Rep Vol. (ml) Theoretical time (min) caffeine and the obtained height is linear with a correlation coefficient of Table 3 and Table 4, shows the result for gradient accuracy for channel A and B and channel C and D respectively. The accuracy is calculated by determining the response factor for each percentage change and comparing it with the average response factor. The obtained accuracies at each level are in agreement with the acceptance/refusal criteria for all channels. Line A Line B Line C Line D a b Accu a b Accur a b Accur a b (min:sec racy acy acy (min) (min:sec) (min) (min:sec) (min) (min:sec) (min) ) : : : : : ; : : : : : : : : : : : : : : Table 3: Gradient accuracy results for channel A and B. Channel A Channel B (min) Height 1 Height 2 Height 3 Average a Response Factor b Acc NA Mean a Response factor = Average height/percentage composition of caffeine b Accuracy = Response factor* 100/Average response factor Table 4: Gradient accuracy results for Channel C and D. a Response factor = Average height/percentage composition of caffeine b Accuracy = Response factor* 100/Average response factor Channel C Channel B (min) Height 1 Height 2 Height 2 Ave. Height a Response Factor b Acc NA Mean Auto injector module 2.1. Precision The ability of the injector to draw the same amount of sample in replicate injections is crucial to the precision and accuracy for peak-area or peak-height comparison for 20 Accur acy external standard quantitation. If the variability of the sample and standard being injected into the column is not controlled tightly, the basic principle of external standard quantitation is seriously compromised. No meaningful comparison between the responses of the sample and the
4 standard can be made. The absolute accuracy of the injection volume is not critical as long as the same amount of standard and sample is injected. Table 5 shows the result for the auto sampler precision test and it proves that the auto sampler is precise as the relative standard deviation for the six injections is less than 1%. Fig 1: Chromatogram for gradient accuracy test of channel A and B Fig 2: Graph which shows the relationship between percentage composition of caffeine and height for channel A and B Table 5: Result for the auto sampler precision Replicate Peak area Average STDVE RSD Linearity Most of the automated LC injectors are capable of varying the injection volume without changing the injection loop. Variable volume of sample will be drawn into a sample injection loop by a syringe or other metering device. The uniformity of the sample loop and the ability of the metering device to draw different amounts of sample in proper proportion will affect the linearity of the injection volume. Most of the employed volumes of injection in HPLC are in the range of 5 to 100 µl. However, in this verification test, the auto sampler linearity has been checked at maximum of 50µL which is the installed loop size for the instrument. As table 6 and fig 3 shows, there is good relationship between volume of injection and response (peak area). The correlation coefficient is which is more than the acceptance/refusal criteria, Table 6: Results for Auto injector linearity with its acceptance/refusal criteria Inj. vol (µl) Peak Area 1 Peak area 2 Peak area 3 Average RSD Correlation coefficient, r Accepted/Not Accepted Accepted Fig 3: Graph for the relationship between volume of injection and peak area 2.3. Carry over Small amounts of analyte may get carried over from the sample injected before and lead to the contamination of the next sample to be injected. The carryover will affect the accuracy of the quantification of the next sample. The carryover assay was carried out by injecting a blank (methanol) after caffeine standard solution (0.2 mg-ml -1 in methanol). The level of carryover was determined by calculating the ratio between the responses (peaks-area) of the caffeine found in the methanol sample and the standard solution. As shown in Table 7, the average percent carry over (n=3) for the auto sampler is 0.26% which is much lower than the acceptance/refusal criteria, 1%. The chromatogram for the sample and blank are shown in fig. 4 and 5. And hence we can say that the cleansing procedure for the auto sampler is good. Moreover, results for precision and linearity prove that the auto sampler is in good condition to use it for quantitative analysis. Table 7: Carry over test results for the auto injector Replicate Peak Area Carry Accepted/Not over Accepted Sample Blank Accepted Accepted Accepted Mean Accepted RSD
5 3. Detector 3.1 Wavelength accuracy Wavelength accuracy is defined as the deviation of the wavelength reading at an absorption or emission band from the known wavelength of the band. The accuracy and sensitivity of the measurement will be compromised if there is a wavelength accuracy problem. Fig 4 shows the UV spectrum for caffeine solution in the range of 190 to 380nm. The obtained λ max and λ min of this spectrum prove that the detector has no problem regarding to wavelength accuracy. Table 8: Summarized results for detector linearity test Conc. Peak Peak Peak Mean RSD (mg/ml) area1 area2 area3 area Correlation coefficient, r = Accept/Not Accept Accepted Fig 5: Graph for Detector linearity Fig 4: UV spectrum for caffeine 3.2 Detector response Linearity The detector linearity is very important when the purpose of work is to carry out quantitative analysis. The linearity of the detector is important to the accuracy for the peak area and peak height comparison between standards and samples and accordingly to the determination of analyte (s) in these samples. Results for detector linearity test have been summarized and graphed in Table 8 and figure 5 respectively. 4. Column Oven Capacity factor, k of an analyte decreases with as temperature increases, and hence the retention of the analysis decreases with temperature [16, 17]. The ability to maintain an accurate column temperature is highly essential to achieve the desired retention time and resolution requirements in the separation process. The temperature accuracy of the column oven is evaluated by placing a calibrated thermometer in the column compartment to measure the actual compartment temperature. As table 9 shows, the column oven is not accurate for temperature more than 50 o C and hence it is highly recommended not to use this HPLC for chromatographic conditions which require column temperature more than 50 o C. Table 9: Results for performance verification of column oven Set Temperature ( o C) Reading ( o C) Correction factor ( o C) Experimental Temperature ( o C) Conclusion The performance of an HPLC system can be evaluated by examining the key functions of the various modules that comprise the system. The common HPLC performance attributes that must be qualified are pump, auto injector, column oven and detector. The obtained verification results for all of the attributes are complying with the acceptance/refusal criteria values as per Herman Lam suggestion. However, the column oven is out of the acceptance/refusal criteria for temperature more than 50 o C. And hence, it is quite logical to use this HPLC instrument for the day to day analytical purpose 1. Prichard, E.; Barwick, V.; Quality Assurance in Analytical Chemistry ; Willey Sommer, K.; Chappel, S.; Kochsiek, M. Calibration and Verification: Two Procedures Having Comparable Objectives and Results ; OIML BULLETIN; vol. XLII, number 1, European Association of National Metrology Institutes (EURAMET); Metrology in Short ; third edition; 22
6 Compañó Beltrán, Ramón; Ríos Castro, Ángel. Garantía de la Calidad en los Laboratorios Analíticos ; Síntesis. 5. Huber, L.; Validation and Qualification in Analytical Laboratories ; Informa Healthcare; B. Palanikumar, A. Thenmozhi, D. Sridharan (2010). A RP-HPLC method for the simultaneous estimation of ceftriaxone sodium and sulbactam sodium injection dosage form. International Journal of Pharmacy and Pharmaceutical Sceiences, Vol 2, A. Narendra, D. Deepika, M. Mathrusri Annapurna (2012). Validated LC method for the estimation of dorzolamide HCl (Carbonic Anhydrase inhibitor) in ophthalmic solutions. E-Journal of chemistry, 9(3), B. P. Nagoria, Amit Maru, Pankaj Muysunic, Subhash Gupta (2011). Method development and its validation for the simultaneous estimation of timolol maleate and dorzolamide HCL in API and ophthalmic solution dosage form by RPHPLC. Journal of Chemical and pharmaceutical Research, 3(4), P. Salo-vaanane, P. Ollilainen, P. Mattila, K. Lehikoinen, E. Salmela-Molsa, V. Piironen (2000). Simultaneous HPLC analysis of fate soluble vitamins in selected animal products after small-scale extraction. Food chemistry, Vol. 71, Macrae (2007). Applications of high pressure liquid chromatography to food analysis. International Journal of food science and Technology, Vol. 15, A. steppuhn, F.L. Wackers (2004). HPLC sugar analysis reveals the nutritional sate and the feeding history of parasitoids. Functional Ecology, 18, Pai-Wen Wu Chieu-Chen Chang, Shin-Shou Chou (2003). Determination of formaldehyde in Cosmetics by HPLC method and Acetylacetone. Journal of food and Drug Analysis, Vol. 11, Wei-Sheng Huang, Cheng-Chinlin, Ming-Chauan Huang, Kuo-Ching Wen (2002). Determination of α- Hydroxyacids in Cosmetics, Journal of Food and Drug Analysis, Vol. 10, Chan, C. et al; Analytical Method Validation and Instrument Performance Verification ; Wiley- Interscience; Guidelines for Calibration in Analytical Chemistry, Part 1: Fundamentals and Single Component Calibration, IUPAC Recommendation, Pure and Applied Chemistry; vol. 70; No. 4; pp , R. G. Wolcott and J. W. Dolan, Column temperature effects in gradient elution, LCGC, 16(12), 1080, J. W. Dolan, The important of temperature, LC-GC, 20(6), 524, Source of support: Nil; Conflict of interest: None declared 23
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