Development and validation of HPLC-DAD methodology for simultaneous qualitative and quantitative determination of thirteen substances with a steroid structure

Figures & data

Figure 1. The target AAS included in nutritional supplements for body building.

Table 1. Preparation of standard stock solutions in methanol.

No.	Standard substance	m, mg	V, mL
1	Testosterone	25.6	25.0
2	Testosterone propionate	25.2	25.0
3	Testosterone enanthate	27.0	25.0
4	17-alpha-Methyltestosterone	26.5	25.0
5	19-Nortestosterone	24.9	25.0
6	19-Nortestosterone 17-propionate	26.4	25.0
7	19-Nortestosterone 17-decanoate	25.9	25.0
8	Methandrostenolon	28.6	25.0
9	4-Androstene-3,17-dione	28.0	25.0
10	Trenbolone	25.1	25.0
11	Trenbolone-acetate	24.8	25.0
12	Trenbolone enanthate	27.0	25.0
13	Boldenone undecylenate	24.9	25.0

Table 2. Elution conditions.

Time, min	Mobile phase A, %	Mobile phase B, %
0–4	100	0	isocratic elution
4–24	100→60	0→40	linear gradient
24–53	60→0	40→100	linear gradient
53–55	0	100	isocratic elution
55.01–60	100	0	isocratic elution

Figure 2. Chromatogram of a mix of targeted compounds at λ = 254 nm, where 13.229 is trenbolone, 13.784 is nandrolone, 14.485 is androstenedione, 16.426 is methanedienone, 19.613 is testosterone, 25.020 is methyltestosterone, 26.073 is trenbolone acetate, 34.209 is nandrolone propionate, 36.748 is testosterone propionate, 46.508 is trenbolone enanthate, 49.498 is testosterone enanthate, 52.780 is nandrolone decanoate and 54.477 is boldenone undecylenate.

Table 3. Coefficient of determination R² and RSD.

No.	Standard compound	R^2	RSD, %
1	Testosterone	0.999	0.221
2	Testosterone propionate	0.999	0.552
3	Testosterone enanthate	0.998	0.371
4	Methyltestosterone	0.999	0.245
5	Nandrolone	0.996	0.964
6	Nandrolone propionate	0.999	0.535
7	Nandrolone decanoate	0.998	0.256
8	Methandienone	0.995	0.852
9	Androstenedione	0.995	0.933
10	Trenbolone	0.999	0.774
11	Trenbolone acetate	0.999	0.244
12	Trenbolone enanthate	0.999	0.306
13	Boldenone undecylenate	0.999	0.261

Table 4. Linearity range for the target compounds.

No.	Standard compound	Linearity range, µg/mL
1	Testosterone	10.24–122.88
2	Testosterone propionate	10.08–120.96
3	Testosterone enanthate	10.8–129.6
4	Methyltestosterone	10.6–127.2
5	Nandrolone	9.96–119.52
6	Nandrolone propionate	10.56–126.72
7	Nandrolone decanoate	10.36–124.32
8	Methandienone	11.44–137.28
9	Androstenedione	11.2–134.4
10	Trenbolone	10.04–120.48
11	Trenbolone acetate	9.92–119.04
12	Trenbolone enanthate	10.8–129.6
13	Boldenone undecylenate	9.96–119.52

Figure 3. Chromatogram of a standard solution—a mixture of all 13 target compounds obtained by optimizing the chromatographic conditions.

Table 5. Method repeatability, precision and recovery of HPLC-DAD procedure for simultaneous quantification of 13 target AAS.

No.	Standard compound	RSD, %	Recovery, average
1	Testosterone	1.58	102.69
2	Testosterone propionate	0.48	101.96
3	Testosterone enanthate	0.67	102.77
4	Methyltestosterone	1.44	103.50
5	Nandrolone	3.00	101.21
6	Nandrolone propionate	0.55	101.62
7	Nandrolone decanoate	0.84	101.72
8	Methandienone	2.28	102.13
9	Androstenedione	2.99	99.45
10	Trenbolone	1.01	101.90
11	Trenbolone acetate	0.38	104.00
12	Trenbolone enanthate	0.36	103.85
13	Boldenone undecylenate	0.42	101.50

Data availability statement

All data that support the findings reported in this study are available from the authors Z.Z. and D.D. upon reasonable request.