The Molecular Weight Of Each Peak Was Then Obtained As Shown In The Figure Below Dissertation Examples

The study aims to identify the 24 testosterone injections taken from different illicit sources. The analytical investigation quantifying range and linearity of testosterone were based on the HPLC and LC-MS techniques where Sustanon 250® was chosen as a pharmaceutical standard against samples. The advanced performance such as great speed, sensitivity and higher selectivity of HPLC made it preferred choice for separation and identification method. For an accurate quantitative analysis, UV detection approach is combined with HPLC. The main advantage of HPLC method is that it does not need heating of the sample heating which decreases the risk of sample degradation19, 22, 23 To verify the existence of testosterone esters in the injections including any impurity or excipient the LC-MS is run with the similar conditions as in HPLC. The application of MS for qualitative analysis along with LC provides specific and precise identification of nearly identical compounds. 26, 27, 31. Hence, the combination of both techniques results in better identification of compounds14, 26 Optimisation of target substances is made against the reference materials. Under high-pressure, different ratios of liquid mobile phase of methanol-to-water ratio were introduced.31, 32, 35 These phase compositions were 95:05, 90:10, 85:15, 80:20, and 70:30 MeOH:H2O, out of which the composition ratio of 85:15 yielded the most favourable results.
3.1.1 Testosterone Standard
A calibration graph for free T. was obtained. However, the R2 value was not high enough probably due to personal errors during the dilution preparation. Due to the nature of this project, quantitative analysis of T. was not an aim as none of the samples should contain free T. The retention time (R.T.) of testosterone at 85:15 was 2.2 minutes. The correlation coefficient of the calibration graph or R2 values should be higher than 0.950 and but higher than 0.990 is considered satisfactory. Value close to 1 delivers a linear slope that acts as an indicator of high sensitivity. Minute disturbances resulting in any error occurs during the pipetting and dilution of the solutions. 33,36 For deriving the best calibration curve it is suggested to consider the weighting factors, because the coefficient value is often sensitive to weighting. 36
3.1.2. T.P. Standard
T.P. was made in the following concentrations: 4, 6, 8, 10, 100 and 1000 µg/mL. A calibration graph was made after the HPLC analysis. In the calibration graph of T.P., the R2 value is 0.9999, which is higher than 0.950. The value is high enough that determines the linearity of graph and produces a straight line. The slope of the curve demonstrates its sensitivity. Thus the selected concentration of T.P. in the solution is accurate. 33,35, 36
The R.T. of T.P. was 4.5 minutes as illustrated in the figure below.
The presence of T.P. was then confirmed by carrying out LC-MS. The following figures illustrate the mass spectrometry along with the UV absorbance.
The actual molecular weight of T.P. is 344.4 but as a result of the ionisation caused by the MS, the molecular weight of the ionised T. propionate would be 345.4 In the results, the ionisation resulted in a shift of the higher molecular weight of 344.4 to 345.4 for T.P. In the figure 10 a sharp peak can be seen at molecular weight of 345.4443 with 100%. Another high and sharp peak is visible at 711 which do not lie within the range of the reference substance. 34,35 .
Figure 10: LC-MS, T.P. molecular weight.
3.1.3 T.E. Standard
T.E. was made in the following concentrations: 2, 4, 6, 8 and 10000 µg/mL. A calibration graph was made of the first four concentrations after the HPLC analysis. The 10mg/mL concentration was analysed to ensure the linearity. It is excluded from the calibration graph as it is very concentrated compared to the other concentrations, and when included in the calibration graph the other concentrations cannot be seen. For a linear graph the suitable ratios are taken, in this case, the R2 value higher than proposed coefficient value of 0.950. 33,35 The value of R2 for T. P. is showing suitable correlation.
The R.T. of T.E. was 13.2 minutes as illustrated in the figure below. However, on most of the samples the R.T. was 13.1 minutes. In LC-MS, fragmentation is produced due to collision provoked dissociation. The esters of testosterone are dependent on the basic molecules and the ester structure of testosterone plays a significant role in its performance. The long ester chains mean the longer activation period. Thus, the dissociation of the molecules is specific towards each testosterone relying on its ester fragments.31 The difference of retention time indicates the difference in molecular weight of the testosterone derivatives.
The presence of T.E. was then confirmed by carrying out LC-MS. The following figure illustrates the mass spectrometry along with the UV absorbance.
The above figure shows the best sharp peak at 401.77 that illustrates molecular weight for T.E. with a slight change that may be due to ionisation during MS. MS technique apparently identifies T.E.
3.1.4. T. A. and T.I Standards
As there was not enough quantity for quantitative analysis of T.A. nor T.I., an unknown quantity of each was analysed to obtain the RT. Each which would be helpful in the identification of T.A. and T.I. in illicit samples. The R.T. of T.A. and T.I. were 3.5 and 9.2 minutes respectively as illustrated in the figure below.
3.1.5. Sustanon® 250
Sustanon® 250 was first analysed to identify the retention time of each component, and then T.P. was quantitatively analysed to ensure that it is within the range. As a result of the lack of standards for quantitative analysis Sustanon® 250 was made up with different dilutions. Each component had a calibration graph and the equation obtained was used to quantify each ester in the illicit samples.
Before the analysis was carried out, a trial run of different mobile phase compositions was carried out. The analysis was done as Sustanon® 250 unlike the previous standards contains more than one ester, so separation is really important. Therefore, Sustanon® 250 required finding the most suitable composition that would result in clear separated peaks, as well as a short RT. The compositions were 95:05, 90:10, 85:15, 80:20, and 70:30. The best composition that eluded the best retention peaks was found to be 85:15.
The analysis was carried out at 245 nm, the wavelength was chosen according to a 3D plot obtained from Sustanon® 250 samples that show the highest peaks at about 240-245nm.
3.1.5.1 Qualitative Analysis of Sustanon
The R.T. of the T.P. was 4.5 minutes that is the same as when the T.P. standard was analysed, 7.7 minutes was the R.T. of T.PP., 9.2 minutes was the retention time of T.I, which is the same retention time as the T.I standard and 33.9 minutes was the R.T. of T.D. These retention times were at first assumed because of the molecular weight of each ester as the lighter the molecule, the quicker it elutes.31
The presence of each component was then confirmed by running a sample of Sustanon® 250 into the LC-MS system as shown in the figure below.

3.1.5.2 Quantitative analysis of T.P. in Sustanon® 250
Sustanon® 250 was diluted to result in the following concentrations of T.P. 6, 9 and 30 µg/mL.
The average area of the 9µg/ml was 300.5852 mAU*s.
The quantity of T.P. was then calculated using the equation obtained from the T.P. standard (Figure 7), i.e. y= 24.111x + 76.492. The calculation is as follows:

Simplifying

300.5852 = 24.111x + 76.492

Reorder the terms:

300.5852 = 76.492 + 24.111x

Solving

300.5852 = 76.492 + 24.111x

Solving for variable 'x'.

Shift all variables with x to the left, all other terms to the right.
Add '-24.111x' to each side of the equation.
300.5852 + -24.111x = 76.492 + 24.111x + -24.111x
Combine like terms: 24.111x + -24.111x = 0.000
300.5852 + -24.111x = 76.492 + 0.000
300.5852 + -24.111x = 76.492
Add '-300.5852' to each side of the equation.
300.5852 + -300.5852 + -24.111x = 76.492 + -300.5852
Combine like terms: 300.5852 + -300.5852 = 0.0000
0.0000 + -24.111x = 76.492 + -300.5852
-24.111x = 76.492 + -300.5852
Combine like terms: 76.492 + -300.5852 = -224.0932
-24.111x = -224.0932
Divide each side by '-24.111'.
x = 9.294230849

Simplifying

x = 9.294230849
Thus, the 9µg/ml concentration had 9.29µg/ml that is 103.2% of the expected quantity. The concentration complies with the BP requirements. Therefore, it can be understood that all the other esters are within the range and are acceptable to be used as standards to quantify the esters in the illicit samples. The above equation was used to quantify T.P. in the illicit samples.
3.1.5.3 The use of Sustanon® 250 as a standard to quantify T.P., T.PP., T.I. and T.D.
Sustanon® 250 was diluted into the concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 µL/mL. A calibration graph was then obtained for each ester in Sustanon® 250 as shown in the figures below:
The equations obtained from each calibration graph were then used to quantify the esters in the illicit samples. The R2 value for T.PP, T.I., and T.D is similar 0.9998 that is high enough for verifying the accurate sensitivity of the method. It shows the suitable value of these three esters in Sustanon pharmaceutical substance. For the same R2 the difference in coverage area is visible that demonstrate the variation in the amount or weight of the analytes.
3.2 Analysis of expected excipients
LabMax 28 have analysed a pharmaceutical grade Sustanon® 250 samples using GC-MS, and it was found that it contains BA, palmitic acid ME, hexadecanoic acid ethyl ester, oleic acid ME, and ethyl oleate. According to the eMC 29 Sustanon,® 250 contains Arachis oil and BA as excipients. Arachis oil mostly contains oleic and linoleic acids 30.
While going through anabolic steroids users’ forms on the internet, most of them use BB as a solvent to dissolve a high concentration of the raw testosterone esters. Not many of them use BA to preserve the oil-based injections as BA causes burning at the site of the injection. They tend to sterile their preparations in a hot water bath. Mostly they use arachise oil or purchase oleic acid from eBay.com. However, other vegetable oils have also been used such as soybean, sesame and grape seed oil. The excipients that are most commonly used are arachise oil, grape seed oil, rapeseed oil, olive oil, BA and BB. Therefore, standards of them have been prepared.
The MeOH and DCM used in the mobile phase showed peaks at 1.3 and 1.7 minutes respectively as shown in the figures below:

The BA and BB showed peaks at 1.6 and 3 minutes respectively as shown in the figures below:

3.3 Analysis of illicit Samples
Sustanon is the form of testosterone available in the market. This pharmaceutical product contains four derivatives of testosterones namely, T. Propionate, T. Phenylpropionate, T. Isocaproate, and T. Decanoate which have been proved with The results extracted from HPLC and LC-MS. The variation in R.T. has confirmed the different ester sized chains indicating the specific activation period. According to the given listing of content ratio the T.D. is present in highest amount; T.PP and T.I exist in similar amount. The LC-mass spectrograph shows the ideal peaks of four analytes at the ratio of 85:15 at similar distance, which is identical to the selected ratio for the sample. The main objective of the project was only testosterone and its esters, but according to the expected presence of excipients in the Sustanon sample the HPLC was performed to detect their presence. The main excipients are olive oil, archaise oil, grapeseed oil and rapeseed oil, along with BB and BA. The LC-MS of DCM, BA and BB confirmed their presence. The R.T. of BA was higher than BB. When comparing the mass spectrogram of illicit samples with the Sustnon sample drug, it illustrates the presence of T.A., T. P., T.I and T.E. Calibration curve obtained from Sustanon® 250 at different concentration of T.P. illustrated that at ideal concentration of 9.29µg/ml it was exhibiting 103.2% of expected quantity. This result was sufficient to illustrate that the other esters in the standard drug are within the limit and can be set as standard for quantifying the esters of the illicit samples.
The calibration graph covered a big area, with R.T. of 4.5 minutes. The calibration curve of standard T.E. showed comparatively small coverage area with high R.T. Standard T.A. and T.I. showed R.T. 3.5 and 9.2 minutes respectively on unknown quantity. On the basis of above results Sustanon® 250 is used as a standard against T.P., T.PP., T.I. and T.D. and calibration curve were drawn. The ratio of quantity in the illicit samples for these analytes showed similarity as in the Sustanon standard sample. Over all the esters from illicit samples showed identical quantitative values to the sustanon sample components. It suggests that the proposed HPLC a competitive method for quantification of injectable testosterone samples. The MS application with LC, for the detection and re-identification of each testosterone has been highly valuable. In this research, the HPLC and LC-MS have delivered the best analysis.
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