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Stability in organic solvents and identity of relevant degradation products

Link to relevant study record(s)

Reference

Endpoint:

stability in organic solvents and identity of relevant degradation products

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 August 1016 to 30 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline required

Principles of method if other than guideline:

An HPLC method using Charged Aerosol Detection (CAD) for the determination of test item concentration ranging from 10 to 200 mg/L in formulations containing arachis (peanut) oil was validated. In addition, formulations prepared at target concentrations of 10 and 200 mg/L test item were analysed to assess test substance homogeneity and resuspension homogeneity/stability following storage for 6 and 11 days at room temperature.

GLP compliance:

yes

Test substance stable:

yes

Transformation products:

no

RESULTS

- Under the described chromatographic conditions, the retention time of the test substance was approximately 10.8 minutes. 

- Figures 1 to 4 (attached) are typical chromatograms of a calibration standard, a processed QC sample, a processed formulation sample, and a processed vehicle blank sample, respectively. 

- The total analysis time required for each run was 17.0 minutes.

 

SPECIFICITY/SELECTIVITY

- As shown in Figure 4 (attached) (and in contrast to the chromatograms shown in Figure 1, Figure 2, and Figure 3, attached), assay specificity/selectivity was confirmed when HPLC/CAD analysis of processed vehicle samples revealed no significant peaks (with S/N > 10) at or near the retention time for the test substance (approximately 10.8 minutes).

 

ASSAY VALIDATION – CALIBRATION REPRODUCIBILITY

- During each of 3 validation sessions, triplicate calibration standards at 5 concentrations were prepared and analysed. Single injections were made of each calibration standard. The resulting test item peak area versus theoretical test item concentration data were fit to the quadratic function using least-squares regression analysis. 

- The results of the regression analyses were used to back-calculate the corresponding concentrations from the peak area data. As per the protocol, the reproducibility of the calibration curve data was considered valid when 1) the inter-session variability, expressed as RSD, of the back-calculated concentrations at each calibration level was ≤ 10% RSD, except at the lowest calibration level where ≤ 15% was acceptable; and 2) the mean back-calculated concentrations at each calibration level were within ± 10% of the theoretical values (%RE within ± 10%), except at the lowest calibration level where %RE within ± 15% was acceptable. 

- The back-calculated concentrations and the associated intra- and inter-session statistics for the test item assay calibration standards are summarized in Table 1 (attached). The inter-session variability (RSD) of the back-calculated concentrations ranged from 4.9% to 8.2% RSD. The inter-session mean concentrations had %RE values ranging from -1.2% to 1.8%. Based on the stated criteria, the reproducibility of the calibration data was acceptable.

 

ASSAY VALIDATION – PRECISION AND ACCURACY

- During each of 3 validation sessions, triplicate QC samples at 3 concentrations were prepared and analysed. Single injections were made of each processed QC sample. The results of the regression analyses were used to calculate the corresponding concentrations from the QC peak area data. The variability (RSD) of the calculated QC concentration data was used as a measure of assay precision, and the difference between the theoretical and calculated mean QC concentrations (%RE) was used as a measure of assay accuracy. According to the protocol, the precision of the method was considered acceptable when the inter-session RSD of the calculated concentrations at each QC level was ≤ 15%, and the accuracy of the method was considered acceptable when the inter-session calculated mean concentration at each QC level had a %RE value within ± 15%.

- The calculated concentrations and the associated intra- and inter-session statistics for the test item assay QC samples are summarized in Table 2. The inter-session variability (RSD) of the calculated concentrations of each QC sample (precision) ranged from 11% to 14% RSD. The inter-session mean concentrations of the QC samples had %RE values (accuracy) ranging from -2.6% to 0.047%. Based on the stated criteria, the precision and accuracy of the test item assay were acceptable.

 

ASSAY RUGGEDNESS

- Assay ruggedness, as required by the SOP, was successfully demonstrated for this method because at least 2 of the 3 validation sessions were performed by different analysts.

 

ASSAY ACCEPTABILITY

- In addition to the experimental samples, each analytical session consisted of (but was not limited to) calibration standards at 5 concentrations and triplicate QC samples prepared at each of at least 2 concentrations. In this study, the formulations were prepared at test item target concentrations of 10 and 200 mg/mL, and the QC samples were prepared at nominal concentrations of 10.0, 100, and 200 mg/mL. According to SOP, for an analytical session to be considered valid, at least two-thirds of the calculated QC concentrations with at least 1 sample at each concentration had to be 85% to 115% of the nominal QC concentration. All reported results were from analytical sessions that met the acceptance criteria.

 

TEST SUBSTANCE STABILITY IN CALIBRATION STANDARDS

- Calibration standards prepared at 40.0 and 100 µg/mL and analysed were stored at room temperature for 11 days before re-analysis to assess test substance stability. 

- The mean post‑storage concentrations ranged from 99.2% to 111% of the pre-storage values (Table 3), which met the (previously stated) protocol-specified requirement for stability (the mean post-storage concentration was not < 90% of the pre-storage value).

 

TEST SUBSTANCE STABILITY IN PROCESSED SAMPLES

- QC samples prepared at nominal test substance concentrations of 10.0 and 200 mg/mL were processed and analysed. 

- The processed samples were stored at room temperature for 11 days before re-analysis to assess test substance stability. 

- The mean post-storage concentrations were 91.5% and 100% of the pre-storage values (Table 3, attached), which met the (previously stated) protocol‑specified requirement for stability (the mean post-storage concentration was not < 90% of the pre-storage value).

 

TEST SUBSTANCE HOMOGENEITY AND RESUSPENSION HOMOGENEITY ASSESSMENT OF FORMULATIONS

- Duplicate samples from the top, middle, and bottom strata of the formulations prepared on 18 Aug 2016 at target test substance concentrations of 10 and 200 mg/mL were analysed to assess test substance homogeneity. The formulations that remained after sampling were divided into aliquots as would be used for daily dispensation. - Representative aliquots were stored at room temperature for 6 and 11 days, at which time the test substance was resuspended by stirring. Duplicate samples were collected from the top and bottom strata of the aliquots and analysed to assess resuspension homogeneity.

- The results of the homogeneity and resuspension homogeneity analyses are presented in Table 4, Table 5, and Table 6 (attached), with the overall statistics summarised in Text Table 3, Text Table 4, and Text Table 5 (below).

 

TEST SUBSTANCE STABILITY IN FORMULATIONS

- Formulations prepared at target test item concentrations of 10 and 200 mg/mL were analysed on the day of preparation. 

- Portions of the formulations were stored at room temperature before being analysed to assess test substance stability. 

- The stability results are presented in Table 7 and Table 8 (attached), with the mean concentration and percent of time-zero values summarized in Text Table 6 (below).

- The post-storage test substance concentrations ranged from 91.6% to 110% of the pre-storage values, which met the (previously stated) protocol-specified requirement for stability, (the mean post-storage concentration was not < 90% of the pre-storage value).

TEXT TABLE 3: HOMOGENEITY ASSESSMENT OF THE 18 AUG 2016 FORMULATIONS

	Low group (10 mg/L)	High group (200 mg/L)
Mean concentration (mg/mL)	11.5	214
SD	0.76	21
RSD (%)	6.6	9.9
Mean concentration % of target	115	107

 

TEXT TABLE 4: SIX-DAY ROOM TEMPERATURE STORAGE RESUSPENSION HOMOGENEITY ASSESSMENT OF THE 18 AUG 2016 FORMULATIONS

	Low group (10 mg/L)	High group (200 mg/L)
Mean concentration (mg/mL)	10.2	232
SD	1.0	12
RSD (%)	9.8	5.0
Mean concentration % of target	102	116

 

TEXT TABLE 5: ELEVEN-DAY ROOM TEMPERATURE STORAGE RESUSPENSION HOMOGENEITY ASSESSMENT OF THE 18 AUG 2016 FORMULATIONS

	Low group (10 mg/L)	High group (200 mg/L)
Mean concentration (mg/mL)	9.14	129
SD	0.92	8.2
RSD (%)	10	6.3
Mean concentration % of target	91.4	64.7

 

TEXT TABLE 6: TEST SUBSTANCE STABILITY IN FORMULATIONS

	Storage duration	Mean concentration in mg/mL (% of time zero) for Low group (10 mg/mL)	Mean concentration in mg/mL (% of time zero) for high group (200 mg/mL)
Room temperature	6 days	10.5 (91.6)	199 (92.9)
Room temperature	11 days	12.2 (106)	237 (110)

Conclusions:

Test item formulations prepared at target concentrations of 10 and 200 mg/mL in arachis (peanut) oil met the protocol-specified acceptance criteria with relative standard deviation for the mean concentration ≤ 10 % at a concentration within the acceptable limits (85 % to 115 % of target). Assessment of test substance resuspension homogeneity and stability in formulations prepared at target concentrations of 10 and 200 mg/mL met protocol-specified acceptance criteria following storage for 6 and 11 days at room temperature (relative standard deviation for the mean concentration was ≤ 10 % and post-storage concentration was not less than 90 % of the pre-storage value).

Executive summary:

METHODS

An HPLC method using Charged Aerosol Detection for the determination of test item concentrations from 10 to 200 mg/L in formulations containing arachis (peanut) oil was validated. Test substance stability was also assessed in calibration standards and processed QC samples stored at room temperature for at least 11 days. Finally, formulations prepared at target concentrations of 10 and 200 mg/L test item were assessed for homogeneity plus resuspension homogeneity/stability after room temperature storage for 6 and 11 days.

 

RESULTS

The test item assay procedure was validated with 3 validation sessions. Quantitation was performed using calibration standards ranging in test substance concentration from 40.0 to 100 µg/mL. The mean back-calculated standard concentrations had inter-session variability ranged from 4.9% to 8.2% relative standard deviation (RSD) and percent relative error (%RE) ranged from -1.2% to 1.8%, which met the protocol-specified acceptance criteria for calibration standards (RSD ≤ 10% and %RE within ± 10% except at the lowest level where RSD ≤ 15% and %RE within ± 15% were acceptable). Assay precision and accuracy were verified by the analysis of QC samples prepared at 10.0, 100, and 200 mg/mL. The mean calculated QC concentrations had inter‑session variability (precision) ranging from 11% to 14% RSD and %RE (accuracy) ranging from -2.6% to 0.047%. The results met the protocol-specified acceptance criteria for precision and accuracy (RSD ≤ 15% and %RE within ± 15%).

 

The test substance in calibration standards and processed QC samples stored at room temperature for 11 days met the protocol-specified acceptance criteria for stability (the post-storage concentration was not < 90% of the pre-storage value).

 

Test item formulations prepared at target concentrations of 10 and 200 mg/mL met the protocol-specified acceptance criteria with relative standard deviation for the mean concentration ≤ 10 % at a concentration within the acceptable limits (85 % to 115 % of target). Assessment of test substance resuspension homogeneity and stability in formulations prepared at target concentrations of 10 and 200 mg/mL met protocol-specified acceptance criteria following storage for 6 and 11 days at room temperature (relative standard deviation for the mean concentration was ≤ 10 % and post-storage concentration was not less than 90 % of the pre-storage value).

Description of key information

Formulations prepared at target concentrations of 10 and 200 mg/mL met the protocol-specified acceptance criteria with relative standard deviation for the mean concentration ≤ 10 % at a concentration within the acceptable limits (85 % to 115 % of target). Assessment of test substance resuspension homogeneity and stability in formulations prepared at target concentrations of 10 and 200 mg/mL met protocol-specified acceptance criteria following storage for 6 and 11 days at room temperature (relative standard deviation for the mean concentration was ≤ 10 % and post-storage concentration was not less than 90 % of the pre-storage value).

Additional information

METHODS

An HPLC method using Charged Aerosol Detection for the determination of test item concentrations from 10 to 200 mg/L in formulations containing arachis (peanut) oil was validated. Test substance stability was also assessed in calibration standards and processed QC samples stored at room temperature for at least 11 days. Finally, formulations prepared at target concentrations of 10 and 200 mg/L test item were assessed for homogeneity plus resuspension homogeneity/stability after room temperature storage for 6 and 11 days.

 

RESULTS

The test item assay procedure was validated with 3 validation sessions. Quantitation was performed using calibration standards ranging in test substance concentration from 40.0 to 100 µg/mL. The mean back-calculated standard concentrations had inter-session variability ranged from 4.9% to 8.2% relative standard deviation (RSD) and percent relative error (%RE) ranged from -1.2% to 1.8%, which met the protocol-specified acceptance criteria for calibration standards (RSD ≤ 10% and %RE within ± 10% except at the lowest level where RSD ≤ 15% and %RE within ± 15% were acceptable). Assay precision and accuracy were verified by the analysis of QC samples prepared at 10.0, 100, and 200 mg/mL. The mean calculated QC concentrations had inter‑session variability (precision) ranging from 11% to 14% RSD and %RE (accuracy) ranging from -2.6% to 0.047%. The results met the protocol-specified acceptance criteria for precision and accuracy (RSD ≤ 15% and %RE within ± 15%).

 

The test substance in calibration standards and processed QC samples stored at room temperature for 11 days met the protocol-specified acceptance criteria for stability (the post-storage concentration was not < 90% of the pre-storage value).

 

Formulations prepared at target concentrations of 10 and 200 mg/mL met the protocol-specified acceptance criteria with relative standard deviation for the mean concentration ≤ 10 % at a concentration within the acceptable limits (85 % to 115 % of target). Assessment of test substance resuspension homogeneity and stability in formulations prepared at target concentrations of 10 and 200 mg/mL met protocol-specified acceptance criteria following storage for 6 and 11 days at room temperature (relative standard deviation for the mean concentration was ≤ 10 % and post-storage concentration was not less than 90 % of the pre-storage value).