Page 27 - Shimadzu Journal vol.2 Issue1

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Fig. 5 shows the recovery rates and peak shapes when a dilution 6. Use in Other Applications
trap is used with indomethacin samples likewise spiked with 6.1 Application to Cleaning Validation Isopropylantipyrine
plasma, and large-volume 100 µL, 200 µL, and 500 µL samples are
injected. Even with the 500 µL injection, a recovery rate of almost Cleaning validations for pharmaceutical production equipment
100 % is achieved, and the peak shape is maintained. require high-sensitivity analysis. However, the samples used for
measurement are often dilute organic solvents. As a result,
pretreatment to concentrate samples is required; this can
sometimes lower throughput. In cleaning validations, Co-Sense for
BA enables direct, large-volume injection without concentrating
the samples, which heightens efficiency.
In reversed phase mode, when organic solvents and other solvents
with strong elution properties are injected in large volumes as the 100 µL injected
sample solvent, peak shapes tend to worsen and there may be
problems with retention times. In cleaning validations for 10 µL injected
pharmaceutical production equipment, ethanol and other organic
solvents are often used as the sample solvent, which hinders Fig. 9 Chromatograms for Indomethacin in an IgG Solution
large-volume injection for high-sensitivity analysis. In addition, Fig. 8 Chromatograms for Isopropylantipyrine/Ethanol Solutions
with ordinary trap injections, recovery rates may decrease due to Using Co-Sense for BA The following shows an example of the analysis of
elution of intended components from the trap column. As a result, indomethacin-derived impurities. Fig. 10 shows the chromatogram
Fig. 4 Effect of On-Line Dilution in LC analysis for cleaning validation, the sample is often from an analysis of indomethacin utilizing an ordinary LC system.
concentrated in advance, leading to decreased throughput. Impurities of 0.07 % are detected. Fig. 11 shows the
6.2 Analysis of Free Bodies and Impurities in Antibody-Drug
Conjugates chromatogram from the analysis of a sample mixture of
indomethacin and IgG in a 1:1 molar ratio using Co-Sense for BA.
Isopropylantipyrine According to the FDA, investigational new drug (IND) applications The impact of the antibodies is eliminated, the recovery rate is 100
Isopropylantipyrine
for antibody-drug conjugates (ADC) have increased markedly since %, and a CV of 2.8 % is achieved. In addition, using LC/MS for
2008, and profiles for free bodies and process-derived impurities detection makes it possible to reliably identify the presence of
are also being discussed [14]. drug-derived impurities, even in ADCs.
50 µL injected 100 µL injected
10 µL injected In the analysis of impurities and free bodies contained in ADCs,
10 µL injected deproteinization is required to eliminate high-concentration
Direct Injection Trap Injection antibodies. If deproteinization is performed using organic solvents
or acids as a sample pretreatment, the impact of the sample
Fig. 6 Chromatograms for Fig. 7 Chromatograms for solvent may worsen peak shapes, and enhancing the sensitivity via
Isopropylantipyrine, Isopropylantipyrine, large-volume injections is often difficult. In addition, for samples
with Direct Injection of Trapping Injection with a high organic solvent ratio after deproteinization, the
an Ethanol Solution without Dilution
recovery rate may decrease with the ordinary trap injection
Fig. 5. Stability for Large-Volume Sample Injections method due to the impact of the sample solvent. Furthermore, if
on-line pretreatment (on-line solid phase extraction) is performed
Fig. 6 shows the chromatograms when isopropylantipyrine/ethanol in order to automate the deproteinization, the recovery rate for
solutions are analyzed using direct injection, and when a trapping
5. Method Validation in Bioanalysis the intended impurities may decrease due to antibody and Fig. 10 Chromatogram for Indomethacin Using Normal LC Analysis
injection is used without on-line dilution. In large-volume (50 µL)
At the FDA and EMA, guidance and guidelines are enacted with injections via the direct injection method, the isopropylantipyrine drug-derived impurity interactions.
respect to the validation of methods for analyzing the peak is leading due to the impact of the sample solvent. In With the Co-Sense for BA dilution trap method, components can
concentration of drugs in biological samples (Bioanalytical Method addition, in trap concentration without on-line dilution, elution be trapped reliably, even with large-volume injections, to enhance
Evaluation: BMV). In July 2013, the Japanese Ministry of Health, from the trap column occurs due to the impact of the sample sensitivity, thanks to antibody removal (deproteinization) and the
With Co-Sense for BA, the injected sample is diluted at a constant Labour and Welfare also issued BMV guidelines. The thinking solvent, and the recovery rate decreases. (The peak for a 100 µL suppression of antibody and impurity interactions. Fig. 9 shows
rate. This is performed to suppress interactions between intended behind ISR (Incurred Samples Reanalysis: evaluation of injection does not equal 10 times that of a 10 µL injection.) the chromatograms from an analysis of indomethacin in IgG using
components and proteins in the sample, and to release intended reproducibility for measurement values from actual samples) and In sample injections via the Co-Sense for BA dilution trap, even Co-Sense for BA. The sample is an acetonitrile solution of 1
components bound to proteins. In this way, even components other aspects of the European and American guidelines were with this sort of large-volume injection of organic solvents, the mg/mL IgG and 2.5 ng/mL indomethacin. (The molar ratio of the
with a high binding rate are reliably trapped and concentrated. A introduced, and its importance emphasized. sample is introduced to the trap column while on-line dilution is indomethacin added is approximately 0.1 % that of the
liquid containing a buffer solution and a low-concentration At first glance, Co-Sense for BA appears to be a complicated performed using water or a buffer solution. As a result, reliable antibodies.) The recovery rate when diluted is 99.6 %. However,
organic solvent is used as the dilution solvent. As a result, even system, and there might be qualification-related concerns. trap concentrations are possible even if guard and other generic the recovery rate drops to 72.3 % when not diluted. Utilizing
plasma and serum samples can be injected directly. In addition, However, utilization of this system not only concentrates samples columns are used as the trap column. In addition, the intended on-line dilution breaks the non-covalent bonds, avoiding a
high sensitivity is achieved due to excellent recovery rates and but also reduces matrix effects in MS analysis. As a result, in terms components are introduced to the analysis column by valve decrease in the recovery rate.
peak shapes, even for large-volume injections [6], [7], [9], [10], of the ongoing issue of method validation, the attainment of more switching after concentration in the trap column, so excellent
[12], [13]. reliable results is anticipated. In addition to generally enforced peak shapes can be obtained. The high-sensitivity analysis of
Fig. 4 shows a comparison of recovery rates based on the presence accuracy, precision, reproducibility, and linearity evaluations, both isopropylantipyrine in ethanol is difficult for ordinary systems, but
or absence of on-line dilution, utilizing indomethacin samples between-day and within-day variance have also been evaluated, with Co-Sense for BA, an approximately 100 % recovery rate and
spiked with plasma. With dilution, the recovery rate is almost 100 and excellent results have been obtained [7], [8], [10], [12], [13]. excellent peaks shapes are obtained even with 100 µL injections.
%. Without dilution, the recovery rate falls to approximately 50 %. Fig. 8 shows the chromatograms when isopropylantipyrine/ethanol Fig. 11 Chromatogram for Indomethacin in IgG Using Co-Sense for BA
solutions are analyzed with Co-Sense for BA.

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