“A grey or gray market (sometimes confused with the similar term “parallel market”) refers to the trade of a commodity through distribution channels that are not authorized by the original manufacturer or trade mark proprietor. Grey market products (grey goods) are products traded outside the authorized manufacturer’s channel.” Wikipedia
SARS-CoV-2 Era Work (March 2020 - December 2020)
Having relocated, lost BIOME funding and access to a GCMS or a wet-lab my options for continuing work dramatically changed. I made the decision to focus on creating cheap and user-friendly drug tests for the hormones commonly used in DIY HRT.
My plan is to synthesize gold nanoparticles, and then use dna aptamers to modulate their aggregation to produce a color change upon introduction of Estradiol/Testosterone.
Initial Validation (March-July 2020)
Aptamer Based Colorimetric Sensing
On 05/16/2020, I ordered 100nmols of the 35nt aptamer from this paper on estradiol aptamers which I call Alsa1535. It arrived 05/22/2020.
Alsa1535: 5'- AAG GGA TGC CGT TTG GGC CCA AGT TCG GCA TAG TG -3'
On 05/27/2020, I was able to confirm that Alsa1535 binds to estradiol valerate and estradiol with impressive linearity (R2=0.9975):
The results cannot be in any major part attributed to the absorption of estradiol valerate alone:
Gold Nanoparticle (AuNP) Synthesis 🎵
My protocol (based on this paper from Nature and this paper from ACS) necessarily excludes some steps (eg. pre-treatment of glassware with aqua regia) due to the limitations of working out of my baby brothers room. It, and my revisions are [in this github repository].
The resulting concentration comes out to be 9.8nM (calculation via the Beer-Lambert law). In calculating the concentration, I noticed an interesting absorption spectrum for the raw nanoparticles:
Attempts to scale up the synthesis were sucessful at 50mL and 100mL. The correlation between the absorbance of said batches in the 600-750nm range with the reaction size is interesting. The dispersal of the Sodium Citrate upon addition to a variable reaction could be affecting particle sizes.
Interns! (July-October 2020)
Through a BIOME mailing lists, I had two folks join the team. They were wonderful and taught me a lot.
Arthur worked on designing new testosterone aptamers for an AuNP assay. He also identified the most promising already extant testosterone aptamers. We cannot evaluate these aptamers until we have access to testosterone again - a feat complicated by it’s scheduled status.
See Sonya and Arthur’s preliminary write up for all the details: sonya-ahuja-paper.gwynu.dev
Sonya worked on identifying progesterone aptamers in existing literature. She identified PG13T1 and PG13T2 from Alhadrami 2017 as promising candidates. The experimental results suggested that they had good binding and selectivity but were ultimately inconclusive.
See her and Arthur’s preliminary write up for all the details: sonya-ahuja-paper.gwynu.dev
|Alhadrami2017‑PG13||61nt||GCA TCA CAC ACC GAT ACT CAC CCG CCT GAT TAA CAT TAG CCC ACC GCC CAC CCC CGC TGC|
|Alhadrami2017‑PG13C4||11nt||TGG GCG GTG GG|
|Alhadrami2017‑PG13T1||31nt||GCA TCA CAC ACC GAT ACT CAC CCG CCT GAT|
|Alhadrami2017‑PG13T2||26nt||GAT TAA CAT TAG CCC ACC GCC CAC C|
|JausetRubio2019‑P5||101nt||TAG GGA AGA GAA GGA CAT ATG ATA CCT CCG AAG TAT CAT GCG GAG CAT GTC CCG AAT TTC ATT CGT TCT CGT GAC TTG ACT AGT ACA TGA CCA CTT GAG G|
|JausetRubio2019‑P6||89nt||TAG GGA AGA GAA GGA CAT ATG ATA CCT CCG AAG TAT CAT GCG GAG CAT GTC CCG AAT TTC ATT CGT TCT CGT GAC TTG ACT AGT ACA TGA CCA CTT GAG G|
|JausetRubio2019‑T6‑46||46nt||GAA GAG AAG GAC ATA TGA TGT GCC GTG AAT ACA GGC CCT TCT CCG C|
|JausetRubio2019‑T6‑54m||54nt||TAG GGA AGA GAA GGA CAT ATG ATG TGC CGT GAA TAC AGG CCC TTC TCT GCT CGT|
|JausetRubio2019‑T6‑57||57nt||AGA GAA GGA CAT ATG ATG TGC CGT GAA TAC AGG CCC TTC TCC GCT CCG CGT TCC GCT|
|JausetRubio2019‑T6‑60||60nt||TAG GGA AGA GAA GGA CAT ATG ATG TGC CGT GAA TAC AGG CCC TTC TCC GCT CCG CGT TCC|
|MiriamJauseTRubio2019‑T6||86nt||TAG GGA AGA GAA GGA CAT ATG ATG TGC CGT GAA TAC AGG CCC TTC TCC GCT CCG CGT TCC GCT TTG ACT AGT ACA TGA CCA CTT GA|
|Skouridou2017‑T5||86nt||TAG GGA AGA GAA GGA CAT ATG ATT GC GTG GGT AGG AAG GGG CGG TGT GAT CTG AA TCG TTC GAT TGA CTA GTA CAT GAC CA CTT GA|
|Target||Aptamer||Target Kd (nM)||T||P4||E2|
|Testosterone||JausetRubio2019-T6||0.80 ± 0.18||103||1090||4725|
|Skouridou2017-T5||4.0 ± 5.8||NR||NR||NR|
|Skouridou2017-T4||1.2 ± 0.2||NR||NR||NR|
|Skouridou2017-T3||1.8 ± 0.6||NR||NR||NR|
|Skouridou2017-T2||33.2 ± 76.2||NR||NR||NR|
|Alhadrami 2017- PG13||35||NR||NR||NR|
|Alhadrami 2017- PG13C4||NR||NR||NR||NR|
|JausetRubio2019-P5||2.03 ± 3.24||1060||112||1373|
|JausetRubio2019-P6||0.57 ± 0.84||1096||183||432|
BIOME Exploration (Summer 2019-Winter 2020)
“BIOME is a student organization dedicated to providing open space for anyone interested in pursuing bioengineering and biology inspired projects.” BIOME.
The funded goals were:
- Validation of UV-Vis as a cheaper alternative to GCMS
- A parallelizable / fast preparation protocol for tablets with varying coatings, sizes and shapes
- Validation and generation of a sub 15 minute GCMS procedure for general purpose quantitation of estradiol in samples
Validation of UV-Vis
UV-Vis was more difficult to get consistent accurate results than previous experiments would suggest. On top of that, prepping UV-Vis samples was a hassle - there were too many steps, and each sample required manual runs without batching.
After much work, I got consistently high R2 values (~0.93-0.97 range) but I kept getting strange nonlinear like responses that set me on edge. At this point, I set UV-Vis aside because there was a) a distinct lack of faith in my data, b) no way the procedures I was using would scale to 100s of samples and c) little hope for a frameshift change in preperation methods.
As time passed, I began to forget the pain of sample prep and again became curious about the source of the non-linearities of my data. Foreshadowing of times to come? Possibly.
Tablet Prep. Protocol
Tablet preparation is important. If I’m going to run 100s of samples, I can’t use my original procedure of:
- Weight tablet
- Powder pill in mortar and pestle
- Weigh by transfer produced powder (~80% transfer is average)
- Wash and dry mortar and pestle
it was too time consuming and rife with potential human error. None of the preparation methods worked at fully dissolving all tablets - with sugar (sucrose) coated tablets being the hardiest of them all.
GCMS Procedure Development
My lab notes:
Initial Quantification Project (Spring 2019)
In Spring of 2019 I enrolled in an Analytical Chemistry Laboratory which had a project component (which was the best part of the class imo). I, along Julia Tanzo worked on developing a quick and versatile method of quantitation of Estradiol in grey market tablets.
Below is the content we presented at the end of the class. The original poster is also available as a pdf poster.
Warning: I am unable to replicate the analysis presented in this poster. See this page for details. In short, there is an off-by-ten discrepancy that I do not have an explanation for. My partner had the flu and we had but a week to create the method and collect data - please be cautious about making conclusions from these data.
After completing this project, I found this paper which also deals with UV quantification of estradiol.
Determination of Estradiol Valerate Content in Grey Market HRT Tablets by Uttmark, Tanzo (Chem 134):
- Estradiol is a feminizing drug used in Hormone Replacement Therapy (HRT) (with a variety of prodrugs1, one of which is Estradiol Valerate which we consider here)
- Hormone Replacement Therapy (HRT) is a regimen involving the administration of “sex hormones” for, among other aims, aligning secondary sexual characteristics, mental health benefits and increased life expectancy.
- Even for individuals that have access to conventional healthcare and can obtain prescriptions for feminizing HRT related drugs, injectable pro-estrogens have experienced shortages in American and European pharmacies2. These factors, among others, have driven transgender individuals, by choice or not, to use grey market alternatives3.
- No investigation or organized effort has ever considered the purity or variability in grey market HRT drugs4.
We aimed to fill this research and humanitarian need by looking at tablet Estradiol Valerate purchased from an accessible venue (eBay) and by developing a general purpose procedure. Here, we determined how much Estradiol Valerate was contained in our samples, the deviation from the reported amount and the variance in these measures across tablets.
Estradiol valerate is a prodrug metabolized by enzymatic hydrolysis into the fatty acid (shown in blue) and Estradiol (shown in black) the active component in feminizing HRT
Progynova tablets presumably containing estradiol valerate were purchased via eBay from gtxl7863 (a now shutdown seller, eBay does not permit the selling of pharmaceuticals) after a brief interview with seven (7) trans individuals who had, or who knew someone that had, procured HRT outside of a doctors prescription to confirm eBay was a reasonable venue for realistic analysis. Each tablet was powdered and dissolved in methanol (methanol and tablet quantification via weighing by difference). The samples were then sonicated at 60℃ for 36 hours to completely dissolve the tablets before filtering through a 20μm filter to remove any debris. The samples were then analyzed primarily via GC MS for quantification and secondarily via UV/Vis and IR spectroscopy to identify future quantification avenues. Samples were run on an Agilent J&W DB-5ht nonpolar column ((5%-phenyl)-methylpolysiloxane, 30m, 0.25 mm. 0.10 µm, 7 inch cage) with a general purpose method consisting of a 7 minute hold at 160℃, a ramp to 260℃ at 10℃/min to hold for 4 minutes and a 10℃/min ramp to 300℃.
This section was not on the original poster. As mentioned above, my off-by-ten attempt to replicate the calculations done here can be seen here.
|Pill||GCMS Area||Concentration (mg/mL)||Powdered Pill in Sample (g)||Initial Pill Weight||Estimated Estradiol Valerate in Pill (mg)||Confidence Interval (mg)|
The raw data is also available here as a csv file.
We quantified ten estradiol valerate tablets. While their reported estradiol valerate content was 1 mg/tablet, we instead found their estradiol valerate content to be on average 1.36 mg/tablet with a variance of 0.0295 mg2. We identified no correlation (R2=0.25) between either tablet weight and estradiol content or peak area. See below for tablet quantification distribution.
We also briefly explored UV/Vis and IR spectrometry as an alternative analysis method as it is both cheaper and faster than GCMS. While IR showed no peaks for quantification of an estradiol standard, UV/Vis spectroscopy did, with a peak at 289 nm having a linear dependence on solution concentration (n=4, R2=0.9958). The same absorbance peak was not identified in estradiol valerate nor did we have enough estradiol samples to analyze a real world example for a proof-of-concept.
Conclusion // Future Work
We have found the estradiol valerate content of grey market HRT tablets to greatly differ from their reported values. While it is still unclear as to the effects of varying dosage on transitioning, it is known that varying estradiol supplements in postmenopausal women increases risk for deep vein thrombosis which is not uncommonly fatal5. For these reasons, it is very important that further work is done to determine the risk associated with “DIY” HRT. We have found that UV/Vis spectroscopy is a promising avenue for continuing quantification of different grey market HRT alternatives that contain estradiol (but not estradiol valerate) without the cost and time associated with GCMS.
This class project was funded by Stanford University and any generated IP is open sourced under a [LICENSE].
B. Duumlsterberg, M. Schmidt-Gollwitzer, and M. Huumlmath-semicolonmpel, “Pharmacokinetics and biotransformation of estradiol valerate in ovariectomized women,” Hormone Research, vol. 21, no. 3, pp. 145–154, 1985 ↩︎
T. U. S. F. D. Administration, “Fda drug shortages.” https://www.accessdata.fda.gov/scripts/drugshortages/dsp_ActiveIngredientDetails.cfm?AI=Estradiol Valerate Injection, USP&st=c ↩︎
C. Nast, “How an estrogen shortage is making life hell for trans femmes.” https://www.them.us/story/estrogen-shortage-estradiol-valerate. ↩︎
Coleman et, al. “Standards of care for the health of transsexual, transgender, and gender-nonconforming people, version 7,” International Journal of Transgenderism, vol. 13, pp. 165–232, Aug. 2012 ↩︎
M. Townsend, H. Jaffer, and L. Goldman, “Adverse health outcomes in transgender people,” Canadian Medical Association Journal, vol. 189, pp. E1046–E1046, Aug. 2017 ↩︎