31
that were only partially separated by IC. Increased sensitivity in impurity detection makes it possible to assess known and unknown impurities more accurately during the early stages of the production processes, reducing the risk of expensive troubleshooting during later stages.
Both MS and sCD detection of mixed organic acids achieved linearity and LOQ below minimum required thresholds
Figure 4. IC-sCD chromatogram obtained for organic acid standards analysed by gradient method at (A) 30°C (black) (B) 40°C (blue) (C) 50°C (pink) (D) 60°C (brown) [2].
better separation of peaks, thereby, offering a greater resolution of components than the isocratic method. The IC-sCD chromatogram and the extracted ion chromatograms of m/z 127 and m/z 171 are shown in Figure 3.
The effect of column temperature on peak shape and resolution
Using the gradient method, the separation of a range of low molecular weight organic standards that could potentially be present as impurities in 2-butynoic acid was investigated. The temperature of the column in IC has previously been reported to influence peak shape in ion-exchange chromatography. Here, the organic acid standards were analysed at column temperatures of 30, 40, 50 & 60°C. The acquired IC-sCD chromatograms corresponding to the different column temperatures are shown in Figure 4. Increasing column temperatures improved the peak shape and reduced peak tailing but resulted in losing resolution on early eluting peaks.
MS detection offered greater selectivity over CD
The organic acid standards, ranging in concentration between 1 ppb and 500 ppb, were analysed using the gradient method and the linearity was evaluated by calculating the R2
0.99 by removing the highest concentration standard when assessing linearity, but that would mean the method’s linearity is only better in the lower concentration range.
The 1 ppb standard was easily detectable for each component with a signal-to-noise ratio of a minimum of 4:1, indicating that the LOQ was less than 1 ppb, apart from pentynoic acid, which had a LOQ of 5 ppb. MS response, as seen in Figure 5, had a greater signal-to-noise ratio than the IC-sCD response and did not have issues with the baseline observed in the IC-sCD chromatogram. Propiolic acid and butynoic acid could not be analysed for linearity or LOQ by IC-sCD in a mixed solution using this method due to partial co-elution. However, these closely eluting analytes differed in their masses, making it feasible to obtain resolution using MS detection, resulting in a LOQ of 1 ppb as shown by the extracted ion chromatogram (EIC) in Figure 5. The additional selectivity of MS detection after IC allowed rapid analysis of peak purity in organic acids and provided quantitation for even those impurities
After obtaining successful linearity and LOQ for all the organic acid standards at low concentrations, it was necessary to confirm whether the linearity and LOQ of these impurities could be achieved in the presence of a 2-butynoic acid sample matrix. Table 2 shows the LOQ and linearity values for each of the individual organic acid components using both IC-sCD and IC-MS in the presence of 25 mg/L 2-butynoic acid. Linearity of response was maintained even in the presence of the 2-butynoic acid matrix. The LOQ obtained using IC-sCD was below the quantification and identification thresholds for impurities as recommended by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) [11], making this technique suitable for routine analysis of all fully resolved impurities. MS, with even lower levels of detection and better sensitivity, can be used for trace analysis.
Relative standard deviation for results across IC with conductivity and MS detection of all organic acids within a 2-butynoic acid sample showed good reproducibility
Impurity testing in the pharmaceutical
Table 3. Repeatability of IC-sCD and MS detection for 50 ppb mixed organic acids and 0.1% w/w organic acid mix in 25 mg/L butynoic acid [2].
r.s.d / % Component values. Table 1 shows the
limit of quantitation (LOQ) and determined linearity for each of the individual organic acid components using both IC-sCD and IC-MS in the order of elution. The R2
values
for the concentration range of 1 to 500 ppb were greater than 0.99 from IC-sCD for most of the acids and greater than 0.95 from the MS. With the MS method for formic acid and propiolic acid, the R2
could be increased to Acetic acid 0.3
Propinoic acid 0.1 Formic acid
0.2
Butanoic acid 0.5 Crotonic acid
1.6
Pentanoic acid 0.5 Butynoic acid n/a Propiolic acid
n/a Pentynoic acid 3.6
Organic acid mix 50 ppb CD
MS
7.9 4.1 9.0 5.3 4.6 5.8 6.8 4.2 5.1
0.4 0.1 0.4 0.8 1.1 0.2 n/a n/a 7.3
At impurity level 0.1 % w/w CD
MS
5.9 5.8 6.4 4.6 5.7 6.0
*Main Peak 5.1 9.8
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