Agilent Development validation of a method for simultaneous determination of paracetamol diclofenac ibuprofen using the Agilent 1120 Compact LC Application Note
Contents
1. 192 2 289 1 772 0 788 load 150 x 4 6 mm 5 pm parti Table 1 cle size column Drug compounds and concentration levels e Agilent EZChrom Elite Compact software a N 80 Benzoic acid ISTD F S 604 lt E 4 S ao o Paracetamol 3 lt 204 Diclofenac Ibuprofen 7 aas a 0 1 2 3 4 5 6 7 8 C Time min j Figure 1 Figure 2 Agilent 1120 Compact LC Analysis of pharmaceutical drugs showing excellent resolution Further the objective of method development was to keep the total analysis time as short as possible The run and equilibration time could be limited to 10 minutes figure 2 The mobile phase con tained trifluoric acetic acid as modifier This influenced positive ly both retention and peak shape The first gradient slope at 4 min utes was fast enough to keep the time between the first and second peaks as short as possible Precision Analyzing drugs with UV detection means that precision of retention times is of utmost importance In addition precision of peak areas must be less than 1 in the low ng range to be compliant with offi cial regulations The precision of retention times and areas was determined using level 2 concen tration The results are shown in figure 3 and table 2 In figure 3 ten consecutive runs were overlaid Limit of detection The limit of detection was calcu lated based on the chromatogram obtained for concentration level 7 see table 3 The limit of detection was in the2. Development and validation of a method i H for simultaneous determination of oe paracetamol diclofenac and ibuprofen e o Using the Agilent 1120 Compact LC o9 e a e Application Note Angelika Gratzfeld Huesgen Patrik Hoerth Daniel Thielsch compet al Sg palem Technologies 1120 Abstract The Agilent 1120 Compact LC is the system of choice for conventional analytical scale liquid chromatography It is an intergrated LC designed for ease of use performance and reliability It is ideally suited for the analysis of pharmaceuticals on account of its capability to achieve high ly precise retention times and peak areas and low detection limits for the analyzed compounds In this Application Note data is presented that demonstrates e Excellent retention time precision lt 0 07 RSD e Excellent area precision lt 1 0 RSD for baseline separated peaks e Excellent linearity with coefficient of correlation gt 0 9999 e Limit of detection LOD 13 298 pg for all compounds analyzed oh Agilent Technologies Introduction For the routine analysis of phar maceuticals compounds and impurities in QA QC it is impor tant to use LC systems that are highly accurate precise and robust The Agilent 1120 Compact LC is based on a proven robust design and delivers the required quality of data This makes it ide ally suited for routine QA QC analysis of pharmaceutical com po
3. low 3 digit pg range for ibuprofen and diclofenac and in the low 2 digit pg range for parac etamol and benzoic acid Linearity Linearity was tested using concen tration levels 1 through 7 Figure 4 shows the linearity of paracetamol as an example The results showed excellent linearity over the entire concentration range Table 4 shows the correlation coefficients for all compounds fee N 401 Benzoic acid ISTD 30 E 8 i S 20 5 2 Paracetamol lt Diclof 10 iclo he 0 L 0 1 2 3 4 5 6 7 8 Ti C ime min J Figure 3 Precision of retention times and areas showing overlay of 10 consecutive runs RSD RSD Injected amount Calculated Ret Times Areas 3 pL injection LOD ng Paracetamol 0 060 0 297 Paracetamol 0 394 0 013 Benzoic acid 0 022 0 179 Benzoic acid 1 145 0 019 Diclofenac 0 020 0 807 Diclofenac 0 394 0 197 Ibuprofen 0 016 0 962 Ibuprofen 0 596 0 298 Table 2 Table 3 Precision of retention times and areas for Limits of detection concentration level 2 N Areas 1 600 000 1 200 000 800 000 400 000 0 0 20 40 60 L Ba A Figure 4 Linearity for paracetamol from 0 886 to 56 7ng per 3 pL injection Correlation coefficient Paracetamol 0 999966 Benzoic acid 0 999954 Diclofenac 0 999975 Ibuprofen 0 999910 Table 4 Correlation coefficients Summary The Agilent 1120 Compact LC was used for the analy
4. sis of pharma ceutical compounds The instru ment was able to analyze these compounds with high precision for retention times and areas The precision for retention times is less than 0 07 RSD and less than 1 0 RSD for areas of baseline separated peaks The limits of detection were between 13 and 298 pg The results showed excel lent linearity over the tested con centration range and the correla tion coefficient was between 0 999910 and 0 999975 Angelika Gratzfeld Huesgen Patrik Hoerth amp Daniel Thielsch are Application Chemists at Agilent Technologies Waldbronn Germany www agilent com chem 1120 in 2010 Agilent Technologies Inc Published June 15 2010 Publication Number 5989 7457EN Agilent Technologies
5. unds In this study the preci sion linearity and limits of detec tion LOD of several pharmaceu tical compounds were evalutated Experimental Chromatographic conditions e Mobile phase A Water 0 05 TFA B ACN 0 045 TFA e Gradient 0 min 25 B 1 min 25 B 4 min 60 B 7 min 70 B e Flow rate 1 5 mL min e Injection volume 3 pL e Column temperature 40 C e Detection wavelength 230 nm Peakwidth gt 0 05 min e Run time 8 min e Post time 2 min Results and discussion Paracetamol ibuprofen and diclofenac were chosen as exam ple compounds for the evaluation of precision linearity and LOD Benzoic acid was used as internal standard ISTD Table 1 lists the concentration levels used The chromatographic method was set up so that all compounds were baseline separated Paracetamol posed a particular problem because it elutes from the C 18 phase column with almost no retention The start conditions were therefore selected so that paracetamol showed at least some retention on the selected column Equipment Compound Ibuprofen Benzoic acid Paracetamol Diclofenac e Agilent 1120 Compact LC com ng 3 pL ng 3 pL ng 3 pL ng 3 pL Level 2 19 075 36 625 28 35 12 6 grated degasser autosampler Shaul 1 d Level 3 9 538 18 313 14 175 6 3 with vial tray column oven an Level 4 4 769 9 156 7 088 3 15 variable wavelength detector Level 5 2 384 4 578 3 544 1 575 see figure 1 Level 6 1
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Agilent Development validation of a method for simultaneous determination of paracetamol diclofenac