Method Article
A protocol for ethanol or isopropanol dosage in hand rub gels
https://doi.org/10.21203/rs.3.pex-1205/v1
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A method of determination of the ethanol or isopropanol content in hydro-alcoholics hand rub solutions is reported. Based on quantitative 1HNMR (q1HNMR), the results are obtained rapidly and accurately. The method is available for viscous solutions to which other simpler techniques do not apply.
As the spread of the COVID-19 pandemic accelerates around the world, many low- and middle-income countries are still struggling to access the diagnostic tests they desperately need to control the disease. Moreover, passive protection methods such as masks and hydro-alcoholic gels are the first means of defence to avoid chaotic spread of the virus among the population. The World Health Organization (WHO) provides a protocol that was taken into consideration in many national legislations.1 In France, at the beginning of the pandemic, we applied this protocol to fabricate several dozens of litres of hydro-alcoholic mixture at our institute and the contents of ethanol or isopropanol in grams per grams of solution was determined by 1H quantitative NMR (qNMR). If the contents of alcohols in liquids gels can be approximatively measured using a simple alcohol-meter, this technic is less appropriate for viscous liquids containing gelling agents. We report here a method of ethanol content measurement using 1H quantitative NMR (1H qNMR). Our intention is to remind the scientific community of this well-known technic.
D2O
3-(trimethylsilyl)-1-propanesulfonic acid di sodium salt (TSPA, CAS: 2039-96-5, 97% purity).
NMR Brucker 400MHz.
3mm NMR tubes (Norell).
Balance (0.01 mg accuracy).
4 mL flat bottom screw neck vial equipped with a centred hole, screw closure closed by a silicone PTFE
syringe (Inject®-F 1 ml)
needle (Henke Sass Wolf, Germany, 0.6X25mm)
Sample preparation:
- Masses are weighed using a Mettler Toledo balance (0.01 mg accuracy) into a 4 mL flat bottom screw neck vial equipped with a centred hole closed by a silicone PTFE (Macherey-Nagel, Germany).
- The standard is weighted first, then 2 mL of D2O are introduced.
- The vial is then closed and gently shaken until all the solid dissolves.
- The gel is added into the vial with a syringe (Inject®-F 1 ml) equipped with a needle (Henke Sass Wolf, Germany, 0.6X25mm) through the silicone join in order to minimize the evaporation of ethanol. - The solution was shaken, then 600 mL were transferred into 3 mm standard NMR tubes (Norell) for analysis.
Pulse Program.
Single pulse, without carbon decoupling (‘zg’ with 90° pulse). Data Points (acquired): 64 K. NS=64. Relaxation delay: D1=60s. Acquisition Time: 4s. Spectral window for proton: SW=30ppm and O1: 7.5 ppm (the sample Temperature is fixed at 19 °C ± 0.1 K).
Post-Acquisition Processing.
Performed with ACDLABS software (1.2, academic version). Zero Filling: to 256K. Line Broadening: LB = 0.1 Hz. Phasing: manually. Baseline Correction: 6th order polynomial (for each signal measured a ratio signal/noise>100 is verified).
It is important to use a screw neck vial closed by a silicone PTFE and an appropriate syringe and needle to minimize ethanol evaporation during the sample preparation.
Sample preparation: 10 minutes
NMR acquisition: 1h
NMR processing: 10 minutes
Quantitative NMR is a valuable method because the measured signals represent a direct measurement of the composition if correct inter-pulse delay are adequately chosen.2 Therefore, in presence of an internal reference of known purity, the composition of a sample can be determined accurately using a single spectrum/ integration sequence (so-called absolute quantification). For this purpose, we used 3-(trimethylsilyl)-1-propanesulfonic acid di sodium salt (TSPA, CAS: 2039-96-5, 97% purity) as standard (also used as chemical shift reference) and D2O for NMR solvent.
After proper weighting of the standard and the gel, the masses of ethanol or isopropanol contained in the samples are calculated by equations 1:
Masse= [ 15 X 0.97 X M(Alc) X SIntAlc X m(Std) ]/[ 218.3 X n1H(Alc) X SInt(Std) ]
= [ 0.06665 X M(Alc) X SIntAlc X m(Std) ]/[ n1H(Alc) X SInt(Std) ] Eq.1
Where M(Alc), n1H(Alc) and m(Std) are respectively the molecular mass, the number of integrated proton and the weighted masse of the chosen alcohol; SInt(Alc) and SInt(Std) being the sum of the integrals for the chosen alcohol and the standard; 15 and 218 and 0.97 being the total number of proton, the molecular mass and the purity of the standard.
For ethanol and isopropanol respectively, the equation resumes to:
Mass(ethanol)=[ 0.615 X SInt(Alc) x m(Std) ]/ SInt(Std) Eq. 2
Mass(isopropanol)=[ 0.572 X SInt(Alc) x m(Std) ]/ SInt(Std) Eq. 3
We found 75% and 68% for handrub solutions produced according to the WHO protocols. The weighted masses were TSPA, 18.14mg, ethanolic gel: 34.69mg (Figure 1) and TSPA: 41.85mg, isopropanol gel: 25.95mg (Figure 2).
1. https://www.who.int/gpsc/5may/Guide_to_Local_Production.pdf?ua=1
2. Pauli, G.F.; Chen, S.N.; Simmler, C.; Lankin, D.C.; Gödecke, T.; Jaki, B.U.; Friesen, J.B.; McAlpine, J.B.; Napolitano, J.G. Importance of purity evaluation and the potential of quantitative ¹H NMR as a purity assay. J Med Chem., 57, 9220-31 (2014).
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