Agilent 5990-3528EN- Running fast LC within USP limits Case study with USP pravastatin sodium chromatographic purity method using the Agilent 1200 Series Rapid Resolution LC system Application Note
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1. N w Be oa a e gt o e N Pravastatin TMBA Related Compound A 1 8 1 9 roo a a ke a Po ep il 2 21 2 2 2 3 2 4 Time min Figure 2 Overlay of three replicate injections of the system suitability sample RCA in pg mL Level impurity S N ratio 1 0 25 0 05 11 8 2 0 5 0 10 25 0 3 0 7 0 14 32 3 4 0 9 0 18 34 9 5 1 0 0 20 38 6 6 1 1 0 22 43 3 Table 3 RCA spiked at levels below and above the limit concentration of 1 0 pg mL The spiked level with a concentration of 0 25 g mL that corresponds to 0 05 is the limit of quantification LOQ for the fast LC experiment Figure 3 shows the overlaid chromatogram at each of the injection levels A uniform rise in the injection amount is seen with the increase in the level of spiked sample The linearity plot of the injection level is shown in Figure 4 where the correla tion coefficient R2 for the linearity is 0 998 Norm 41 3 1 i ar pth fbf belt toh el hab tom pf op oh oP eo he 2 18 2 19 22 2 21 2 22 2 23 2 24 C Time min J Figure 3 The linearity of spiked sample of RCA at spike levels of 0 25 pg mL L1 to 1 1 pg mL L6 a 8 7 6 5 Ey lt E4 2 z LOD i 24 1 am 0 T T T T T 1 0 0 2 0 4 0 6 0 8 1 0 1 2 Concentration pg mL L Figure 4 The graph of linearity solution for RCA shows that the concentration2. 3 M Woodman Improving the Effectiveness of Method Translation for Fast and High Resolution Separations Agilent Application Note Publication number 5989 5177EN 2006 www agilent com chem rrlc Agilent Technologies Inc 2010 Published June 15 2010 Publication Number 5990 3528EN ORE Agilent Technologies
3. of 0 25 pg mL is the concentra tion closest to the limit of detection LOD Conclusion Validating analytical methods can be less time consuming if run time is reduced without compromising chro matographic properties Fast runs con tribute to cost savings in routine quality assurance quality control A faster 8 minute pravastatin chromatographic method was determined from the origi nal 30 minute USP pravastatin chro matographic purity method This faster method was initiated by modifying only those parameters that were permitted in recent USP adjustments for chro matographic parameters The new gra dient conditions were obtained from Agilent Method Translator and were used without any modifications This new fast method meets all the system suitability requirements The resolution between RCA and pravastatin was 3 8 which is well above the NLT 2 0 limit Precision test with standard solution obtained an RSD of 1 0 which is less than the 10 acceptable limit A detec tor linearity test for the fast method obtained an R value of 0 998 The fast method obtained by modifying only within the USP limits shows that the cost per sample is reduced by more than three times References 1 U S Pharmacopeia 31 NF 26 second supplement 2008 2 M Frank Achieving Faster Analysis with the Agilent 1200 Series Rapid Resolution LC System and 2 1 mm ID Columns Agilent Application Note Publication number 5989 4502EN 2006
4. Author Syed Lateef Agilent Technologies Bangalore India Running fast LC within USP limits Case study with USP pravastatin sodium chromato graphic purity method using the Agilent 1200 Series Rapid Resolution LC system Application Note Manufacturing QA QC Abstract Recent revisions in United States Pharmacopeia USP general chapter lt 621 gt allow for adjustments to be made in monographs to enhance the quality of the chro matogram in meeting system suitability requirements These adjustments can be made use of to produce a fast method utilizing the Agilent 1200 Series Rapid Resolution LC RRLC system and Agilent Method Translator software There are vari ous ways to produce a fast method and one of them is to start with an established method such as a USP method This Application Note shows how to start from a USP method and incorporate the allowed adjustments to produce a fast method that meets system suitability require ments The chromatographic purity test for pravastatin as per the USP method sug gests a 30 minute gradient with a 3 5 pm particle size column and 1 mL min flow rate These chromatographic conditions can be adjusted to use a 1 8 um particle and 1 5 mL min flow rate which will provide faster run times Besides particle size and flow rate column dimensions and column temperature can also be adjusted to pro duce a faster method The transition to the fast method was quickly and effectively achieved by
5. P method and the fast method System suitability of fast run using autosampler SL plus According to the USP a system suitabil ity mixture containing pravastatin TMBA and RCA spiked at the limit con centration of 1 pg mL should show a resolution of not less than NLT 2 0 and RCA should have an RRT of about 1 1 Figure 2 shows the overlay of three replicate injections of the system suit able sample performed by the fast method The results show that RCA has an RRT of about 1 1 and a resolution of 3 8 which meets the system suitability requirements Standard deviation of pravastatin peak area at low concentration The standard sample containing 1 25 ug of pravastatin TMBA was injected six times to test the RSD of the peak area of pravastatin According to the USP method six replicate injections of the standard sample should have an RSD for the peak area of not more than NMT 10 0 A value of 1 0 was obtained which meets the precision requirement Linearity of spiked impurity in fast run The USP method specifies an upper limit concentration of 0 2 for RCA which is 1 pg mL assuming pravas tatin has a concentration of 0 5 mg mL To test the detector response and deter mine the injector s precision RCA was prepared at a concentration ranging from 0 25 to 1 1 pg mL and spiked into 0 5 mg mL pravastatin sodium solution Table 3 shows the signal to noise S N ratios obtained at each of the spiked levels mAU
6. RRT test sample Pravastatin sodium and four of its impurities 6 epipravastatin RCA pravastatin lactone and com pactin were dissolved in diluent to a concentration of 65 pg mL for pravastatin and 7 5 pg mL for the impurities The RRT test sample was prepared to determine the RRT of each impurity for comparison with values reported in the USP Prava statin lactone and compactin were obtained from Varda Biotech Mumbai India RCA was obtained from the USP and 6 epipravastatin was obtained from the European Pharmacopoeia EP e System suitability sample As described in the USP pravastatin 1 1 3 3 tetramethylbutylamine pravastatin TMBA and RCA were dissolved in diluent to obtain a con centration of 0 6 mg of pravastatin TMBA and 0 001 mg of RCA per mil liliter The system suitability sample was used to determine the value of the RRT of RCA versus pravastatin and the resolution between them Both pravastatin TMBA and RCA were obtained from the USP Standard sample Pravastatin TMBA was dissolved in diluent to a concen tration of 1 25 pg mL The standard sample was used to determine the relative standard deviation RSD of the peak areas Linearity test sample Six concentra tion levels of RCA from 0 25 to 1 10 pg mL were prepared as spiked amounts in a 0 5 mg mL sample of pravastatin sodium Equipment The Agilent 1200 Series Rapid Resolution system included Agilent 1200 Series binary pump wit
7. h degasser Agilent 1200 Series autosampler SL and Agilent 1200 Series autosampler SL Plus with thermostat Both mod ules were used interchangeably in the same LC system Agilent 1200 Series diode array detector Agilent 1200 Series thermostatted column compartment SL Agilent 1200 Series RRLC mode Binary pump in low delay volume configuration without the mixer and damper in the flow path and low delay volume capillaries 0 12 mm id beyond the injection valve were used Selecting a low delay volume reduction in the autosampler did not make any major difference in elution of the peaks Modules were stacked in two parts such that the pump and degasser were separate from the rest of the modules to minimize delay volume as described earlier HPLC mode No delay volume config uration was made in the binary pump and standard green tubing 0 17 mm was used at all points Module stacking was similar to the RRLC mode Agilent Method Translator version 2 0 Parameters Detail Wavelength for DAD 238 nm BW 4 Ref 300 20 Column USP method Agilent ZORBAX SB C18 4 6 mm x 75 mm 3 5 um Fast method Agilent ZORBAX SB C18 4 6 mm x 30 mm 1 8 pm Diluent 50 50 methanol water Needle wash Flush port 3 sec using diluents Sample temperature 15 C Mobile phase Buffer pH 7 0 0 08 M phosphoric acid solution adjusted with triethylamine to pH 7 0 Buffer A Water buffer pH 7 0 acetonitrile 52 30 18 Buffe
8. r B Water buffer pH 7 0 acetonitrile 10 30 60 USP method Time Buffer B Flow rate 1 0 mL min 0 0 Post time 3 0 min 3 0 0 Column temperature 25 C 26 5 100 Injection volume 10 uL 26 6 0 Response time 2 sec 30 0 Fast method Time Buffer B Flow rate 1 5 mL min 0 0 Post time 0 8 min 0 8 0 Column temperature 35 C 7 07 100 Injection volume 4 0 7 09 0 Response time 1 sec 8 0 0 Results and discussion Retention time of impurity relative to the pravastatin retention time in fast run Agilent Method Translator was used to convert the USP pravastatin chromato graphic method into a fast method The output method from Agilent Method Translator was used without any modi fications The desired flow rate can be input into the translator which calcu lates the respective gradient condition The advantage in using the Agilent 1200 Series RRLC system is that it is interconvertible between HPLC mode and RRLC mode In this Application Note HPLC mode was used to run the conventional USP method and RRLC mode was used in the fast method An RRT test sample containing a mix ture of four impurities along with pravastatin was tested in both HPLC and RRLC modes Figure 1 shows the chromatographs of the RRT test sample tested in both modes A fast chromato graph show adequate separation of all the four impurities in a manner similar to the conventional method The RRT test sample was used to cal culate RRTs and for compari
9. son with the RRT provided in the USP Table 2 shows the comparison and excellent match between the RRTs obtained experimentally and those reported in the USP method The results show that the RRT values as reported in the USP are about the same as those obtained by the fast method fo y mAU 5 1 A S n 175 J g J a 150 4 125 J x 7 o 5 e S 100 3 siz al oO a s 75 J s 5 E ae F J Lu Ss D 9 50 J x g 2a wo T 3 a cc e 25 1 0 To 5 7 5 10 12 5 15 17 5 20 22 5 25 Time min mAU 1 B 175 J 5 J n A J gt 150 4 1 a 125 q lt o 3 T 100 4 S 2 1 al 3 75 3 J 8 5 J a 50 4 iS T 1 amp gt J S 25 4 oc a E T T T T T T T T 1 2 3 4 5 6 Time min Ne 4 Figure 1 A Pravastatin chromatogram performed as per the USP method B Pravastatin fast method based on Agilent Method Translator Experimentally Experimentally obtained USP method obtained fast method USP reported 4 6 mm x 75 mm 4 6 mm x 30 mm Name RRT 3 5 um column 1 8 pm column 3 Hydroxypravastatin 0 33 Rail 6 Epipravastatin 0 92 0 92 0 94 3a Hydroxyisocompactin 1 1 1 1 1 1 Pentanoyl impurity 1 2 T i Pravastatin lactone 1 8 1 8 1 8 Compactin 3 1 3 1 3 2 Impurity not included in the experiment Table 2 RRTs of the four out of six impurities reported in the USP method along with the results obtained experimentally as performed by the US
10. tatin method As per the revised general chapter lt 621 gt the pH of the mobile phase the concentration of the salts in the buffer the ratio of the components in the mobile phase and the wavelength can also be adjusted but were not done so in this experiment Pravastatin sodium has six impurities that are reported by the USP 1 3 Hydroxypravastatin 2 6 Epipravastatin Original pravastatin chromatographic Fast gradient Permitted adjustments method Permitted limits conditions Column length 70 75 mm 22 5 127 5 mm 30 mm Column id 25 4 6 mm 3 45 5 75 mm 4 6 mm Particle size 50 3 5 pm 1 75 pm 1 8 um Flow rate 50 1 0 mL min 0 5 1 5 mL min 1 5 mL min Injection volume 10 uL Variable 4 0 uL reduced until it is consistent Column temperature Not specified Variable 35 C 10 C Value obtained from Agilent Method Translator Table 1 The permittable adjustments in the USP pravastatin chromatographic purity method 3 30 Hydroxyisocompactin also called Related Compound A or RCA 4 Pentanoyl impurity 5 Pravastatin lactone 6 Compactin Only impurities 2 3 5 and 6 were used to demonstrate the fast impurity method on the Agilent RRLC system Experimental Sample preparation e Diluent A 1 1 mixture of methanol and water was prepared The methanol was HPLC grade J T Baker Milli Q water Millipore was used for the experiment e Relative retention time
11. the use of Agilent Method Translator Such fast methods derived from the USP can be used in high throughout environments as the new method is closest to the validated monograph method j Agilent Technologies Introduction Pravastatin sodium helps to reduce cholesterol biosynthesis thereby pre venting cardiovascular disease The USP chromatographic purity test for pravastatin recommends using a L1 column with dimensions of 4 6 mm x 75 mm 3 5 um particle size and 1 mL min flow rate However under the revised general chapter lt 621 gt the USP allows adjustments in the chro matographic parameters to be made but only when adjustments have improved the quality of the chro matogram in meeting system suitability requirements In this Application Note we have taken advantage of the permittable adjustments in the USP to produce a faster method and test sys tem suitability parameters Faster runs are possible by starting with any HPLC method and determining what parame ters are required if choosing smaller id and smaller particle column Further increase in speed is possible by increasing the temperature and flow rate while decreasing the gradient time 2 The faster LC run has the advantage of saving money in terms of solvent con sumption as well as higher productivity in analyzing a larger number of samples Table 1 shows the permittable adjust ments in the chromatographic parame ters and its application in the prava s
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Agilent 5990 3528EN Running fast LC within USP limits Case study with USP pravastatin sodium chroma