Table 2.
Validation of the GC-MS/MS analytical procedure in mouse brain
| Steroid (mol wt) | Detection limit (pg/g) | Accuracy (%) | Intra-assay variation (%) | Inter-assay variation (%) |
|---|---|---|---|---|
| Pregnenolone (316) | 2 | 102.9 | 5.1 | 5.5 |
| P (314) | 20 | 105.9 | 2.2 | 3.6 |
| 5α-DHP (316) | 5 | 101.3 | 3.2 | 4.8 |
| 5β-DHP (316) | 2 | 103.4 | 3.6 | 6.8 |
| 3α5α-THP (318) | 10 | 97.5 | 5.4 | 7.4 |
| 3β5α-THP (318) | 0.5 | 99.6 | 4.5 | 5.6 |
| 3α5β-THP (318) | 10 | 105.1 | 6.2 | 6.5 |
| 20α-DHP (316) | 10 | 103.6 | 8.2 | 9.2 |
| 5α20α-THP (318) | 1 | 106.2 | 4.3 | 5.1 |
| 3α5α20α-HHP (320) | 5 | 98.4 | 3.2 | 3.5 |
| 3β5α20α-HHP (320) | 5 | 99.6 | 2.9 | 3.0 |
| 3α5β20α-HHP (320) | 5 | 95.3 | 2.6 | 2.7 |
| 20α-DHPREG (318) | 1 | 107.1 | 1.5 | 2.5 |
| Testosterone (288) | 1 | 99.8 | 1.1 | 1.8 |
| 5α-DHT (290) | 2 | 101.4 | 3.9 | 4.3 |
| 3α5α-THT (292) | 1 | 102.6 | 3.6 | 4.9 |
| 17β-estradiol (272) | 1 | 96.4 | 1.3 | 1.9 |
| DOC (330) | 5 | 98.2 | 1.5 | 1.7 |
| 5α-DHDOC (332) | 5 | 102.6 | 5.4 | 5.8 |
| 5β-DHDOC (332) | 5 | 104.5 | 6.1 | 6.9 |
| 3α5α-THDOC (334) | 2 | 10.10.4 | 4.9 | 6.1 |
| 3α5β-THDOC (334) | 2 | 98.2 | 3.4 | 4.0 |
| Corticosterone (346) | 20 | 97.6 | 2.5 | 3.0 |
The analytical protocol has been validated for all targeted steroids using 200 mg extracts from a pool of male mouse brains. The limit of detection was determined as the lowest amount of compounds that could be measured by GC-MS/MS with a signal-to-noise ratio >3. The linearity was assessed by analyzing increasing amounts of mouse brain extracts (20, 50, 100, and 200 mg) in triplicate with a coefficient of correlation ranging from 0.992 to 0.999. The accuracy of the assay was evaluated by determining the analytical recovery, which was defined as C/(C0 + S) × 100(%), where C is the concentration of the steroid in the spiked brain extract (200 mg), C0 is the concentration of a steroid in the unspiked brain extract (200 mg), and S is the spiked concentration. The precision of the intra-assays and inter-assays were evaluated by analyzing 5 replicates of 200 mg brain extracts on 1 d and over 4 d, respectively.
Abbreviations of steroids are as for Table 1.