Quality and doses in basic analysis methods

Basics of QC of oil-based parenterals.

Well, everybody knows that steroid based injections are faked. We will pass by the meter why it happens and will discuss how it happens and what could we do to avoid fakes. First of I would like to show you how the oil-based parentals are normally made. After that I’ll try to discover basics of quality testing and some advanced things in brief.

So, lets look on fig 1 which is illustrates the simplified technical process. For the manufacturing required “vehicle” solution which is contains pharmaceutical parental-grade oil with excipients which are suppress bacteria growth and enhances solubility of the API i.e. steroid ester as well as could suppress oxidation of the API. Normally, excipients such as benzyl alcohol are reducing the pain in site of injection, but this is not in the case of fake. I would say faker’s formulation induces pain in the site of injection (I’ll explain later why). The theory of the preparation of the oil-based formulation

is simple. Oil-based vehicle controls water content i.e. blood and lymph access at the injection site to make a long term effect. Excipients like benzyl alcohol (BA) are typically added in amount of 9 mg/ml of formulation, this level is considered to be optimal for the local anesthesia. Fakers some times use BA as a solubility enhancer in order to have better solubility of low-quality API.

The concentration of BA could reach 5% in such formulations. In such dozes BA acts as a cytotoxic agent which is induces local necrosis in the injection site. This is not very dangerous but extremely unpleasant. Being injected not deep (for example 10-15 mm in depth) such formulation causes necrosis between muscle and skin, this can cause serious discomfort and could be dangerous because injection site is continue bleeding thus could be infected. What else? I think that fake formulations do never content anti-oxidation agents. Factory made formulations could contain antioxidant or argon (noble gas) above the liquid. Usually, fakers unable to fill vials with argon during corking.

Now lets discuss oil and API. These two components are added in a significant amount to the formulation, so the quality of them is not unimportant. Oils are widely used in different applications so there are many quality grades of them. Usually oils for pharmaceutical applications are good enough and not hazardous, but being improperly stored or operated oil could be polluted with moisture,

bacteria and sediments.  Usage of non-pharmaceutical oils, used to be rare, since it became more and more difficault to buy pharma-grade oils, and prices of UG products became dirt cheap. UG labs more and more started to use cookingoil from the Supermarket, as our analyses proved. API is produced in different grades as well. There are several grades of quality human-grade for parenterals or for tabs, GMP-grade, animal grade. Some good guys are telling “we use human grade API for the dog formulations” some baones telling “we use animal grade stuff on humans”. All of them are not very hazardous if operated and stored properly. Typically, the main problem of API is the content of free hormone, i.e. testosterone enantate contains more or less free testosterone and some amount of enantic acid. These components are not hazardous, but significant content of the testosterone causes low temperature sedimentation of the drug or turbidity. Fakers are trying to

suppress this effect by overdosing BA. Widespread problem is the presence of moisture in formulation. This is a key feature that allows verify the quality of a drug. Where is the moisture coming from? First of, low quality oil, API stored in refrigerator in an opened can (fakers are usually makes small amounts, so they can not use factory pack at once), and the third path-way ­

through bad stoppers during the sterilization in water-steam autoclave. In general, moister is the enemy number one of oil-based formulations, especially if it is a handicraft drug. Moisture causes hydrolysis of ester, loss of solubility, turbidity and sedimentation of the API. How we do detect moisture?

We can look if the oil in vial is wetting the glass. Low quality mixtures have a poor wetting. We can check if the sediments are present as well. It is easy ­ take a laser pointer, shake vial vigorously and watch for the particles in passing light, use a dark room to find illuminated particles. Sediments are coming to handicraft with oil, API powder, and from ambient air (it could be just a usual dust). Factory made drugs are produced in dust-free rooms, API and oil are stored in sealed cans. Before the packing oil solution is normally filtered through 0,22 mkm filter. It is obviously that factory-made drugs have no sediments.

I discovered basics methods of a fake detection and some ideas where problems are come from and how to find them fast, - the pain in the injection site, moisture detection and sediments detection. These methods are not very reliable. Sure, if you will find the turbidity or sediments it is 99% fake. But sometimes you will be unable to check the drug with these methods. Now I’ll describe I brief advanced techniques and methods of chemical analysis. In general there are two major problems here ­ the dosage and the API kind. It is well-known problem when API is under dosed and if it is substituted with another one, or the both cases in the same time ­ for example vial contains 25 mg/ml of trenbolone acetate and 25 mg/ml of testosterone propionate instead of 50 mg/ml of trenbolone acetate. Here is two kinds of techniques are employed ­ quantitative analysis to determine API content and qualitative analysis to determine the nature of API added.


This type of analysis could be made according USP recommendations. There are two different methods may be employed ­ high-performance liquid chromatography (HPLC) and spectrophotometry in ultra-violet range(UV). Both of methods have disadvantages, so the analysis should be made by qualified specialists.


This method is based on the separation of the drug formulation to individual compounds and quantitative analysis of the target compounds buy UV-detector. For the separation the solution of drug in isopropyl alcohol is injected to a chromatographic column. Different compounds have a different linear migration speed in this column, so they are separated according the specific retention which is considered to be individual to each compound. Normally, long chain esters are not perfectly separated by conventional commercial HPLC instruments. Adaptation of the instrument for the silicagel column is expensive and usually analysis are made in low-cost way using conventional reversed-phase columns C8 or C18.  Thus compounds are moving with different speed we can identify a target ester using migration time. Quantification in HPLC is provided by a UV spectrophotometric detector. USP recommends wavelength 240 nm for the quantification of testosterone esters and nandrolone esters, many others could be detected at same wavelength as well. This wavelength corresponds to the maximum of UV light absorbtion by steroids. Short wavelengths are not recommended due to non-specific absorbtion.

It is believed that HPLC coupled to diode array detector could provide the identification of steroid base. I think that it is not reliable technique, most of steroid bases have similar structure, so they will have a similar UV spectra. Sure, some steroids as trenbolone have unique fingerprint in UV but many others are too similar.

To find a concentration of ester we can add an additional known amount of pure ester. To have a good quantification more then 2 additives are necessary, i.e.if we have a concentration about 50 mg/ml of testosterone propionate we need to analyze this sample we should make a picture of this sample and two pictures with standard additives to the sample solution of testosterone propionate. For the testosterone mixtures such sustanon concentrations of esters could be recalculated to molar concentrations in order to make quantification using single ester as a standard.

UV-Vis spectrophotometry.

This method is well known low-cost way to determine the content of many different compounds by the specific light  absorbtion. This method employees a simple principle ­ the light absorbtion is proportional to the compound concentration. For all chemical compounds there is a wavelength where compound absorbs the light. For most steroids the best wavelength is 240 nm. But it is

virtually impossible to measure concentration of testosterone ester in this region in a drug. This problem caused by benzyl alcohol which is have a strong absorbtion at 240 nm. Benzylbenzoate have similar high absorbtion (by weight). It is possible to reduce concentration of BA by chemical manipulation, but if  formulation contains BB, UV-Vis gives up.

API identification.

There are few methods which are allows to get a fingerprint of the molecular structure. These methods are widely used in chemical analysis, molecular structure research and have a forensic grade reliability due to detailed fingerprint of all molecular parts or atoms. These methods are:

1.      Infrared spectra, or Fourier ­ transform infrared, FTIR. The most suitable for oil formulations technique is FTIR identification, this method allows direct measurement of spectra in the oil solution. FTIR spectrometer coupled to multiple internal reflectance (MIR) accessory allow both ­ fingerprint and quantification of the drug. There is the broad number of manufactures of such equipment: PerkinElmer, Bruker, Thermo-Nicolet, Varian, Shimadzu and many others.

2.      Electron-impact mass-spectra ­ EI/MS, usually coupled to gas chromatography. This method is widely used and named GC-MS. These equipment is widely used, but not the best for this kind of analysis, manufactures are: Thermo, Agilent, PerkinElmer, Shimadzu, Varian and many others.

3.      Nuclear magnetic resonance spectra, NMR, of two kinds ­ proton magnetic resonance (PMR) and C13 isotope magnetic resonance spectra, C13-NMR. This equipment is expensive and made by Bruker,  Hitachi, Varian, Shimadzu.

4.      Liquid chromatography coupled to mass-spectrometer. Well, usual LC-MS just do not gives structural information. This technique shows only molecular weight +

1. Instruments used for the molecular fingerprinting are terribly expensive because of presence of 2 or 3 or even 4 mass analyzers in the instrument.  LC units made by: Waters, Agilent, PerkinElmer, Varian, Shimadzu. MS units made by: Bruker, ABI-Sciex.

Wet chemistry.

Instrument ­ based analysis have there limitations. Concerning oil solutions of long-chain esters these disadvantages are critical. Only FTIR/MIR allow identify ester directly with minimum sample manipulation. Other techniques require sample preparation or even preparation of pure steroid base. At this point professional analysts starts there game. Typical pathways has there target in obtaining of pure steroid ester or steroid base or reducing of interfering effects of benzyl compounds. There are several basic techniques ­ the freezing of oil at low temperature with different additives to cause the precipitation of ester, vacuum evaporation of benzyl alcohol, hydrolysis of a sample in order to have a steroid base. These techniques could be employed one-by-one to have a desired effect.