Investigating the Factors Having the Important Role in the Standardization of Herbal Distillates Methanol Content

Research Article

Austin Environ Sci. 2021; 6(2): 1056.

Investigating the Factors Having the Important Role in the Standardization of Herbal Distillates’ Methanol Content

Rafizadeh A1*, Shariati Sh2, Safarzadeh Vishekaei MN3, Honarchian Masihi P4 and Rafizadeh M4

1Departments of Nursing & Midwifery, Rasht Branch of Islamic Azad University, Rasht, Iran

2Department of Chemistry, Rasht Branch, Islamic Azad University, Rasht, Iran

3Department of Agronomy, Rasht Branch, Islamic Azad University, Rasht, Iran

4Department of pharmacy, Mazandran University of Medical Sciences, Ramsar, Iran

*Corresponding author: Rafizadeh A, Departments of Nursing & Midwifery, Rasht Branch of Islamic Azad University, Poletaleshan, Lakan road, Rasht, Iran

Received: April 15, 2021; Accepted: May 06, 2021; Published: May 13, 2021


Blurred vision leading to nervous blindness is the only symptom of chronic methanol intoxication that occurs following prolonged drinking some kinds of herbal distillates and has been motivated serious anxieties. The methanol content of these products is not usually measured which may be due to the lack of applicable standards in this regard. In this study, the methanol concentrations of some kinds of herbal distillates were quantified by two different methods to define the most effective factors in the determination of methanol permitted dose of these products. In this study, the methanol contents of 60 different samples were examined by a newly designed kit and Gas Chromatography methods, and finally, based on gained results some recommendations were presented about how standardization of herbal distillates’ methanol content. All samples have different concentrations of methanol that in some cases, their methanol content seems enough for the creation of chronic methanol intoxication. Also, access to an appropriate and efficient national standard for controlling of methanol content of herbal distillates seems to be necessary.

Keywords: Chromotropic acid; Herbal distillates; Methanol; Methanol concentration; Methanol intoxication


Methanol is a colorless alcohol with a characteristic smell that has many extensive industrial and household uses [1]. Land plants’ growth and their biologic activities have the most contribution to methanol production in nature [2-7]. This process is mostly formed by degradation of the pectic substances of the middle lamella of plant cells. Pectin degradation is one of the essential stages of plants’ cell walls development during their growth [7-9]. The plants’ cell walls are distinguished into two types based on their own molecular weight that each of them has different potential for methanol production [8,10]. In the presence of Pectin Methylesterase Enzyme (PME), high methoxyl pectin is converted into low one by releasing methanol, that in fruits and vegetables is accompanied with the softness of their cell walls (ripening) [7,11,12]. This alcohol can also produce from enzymatic cleavage of lignin, demethylation of DNA and protein repair pathways [8,10]. Likewise, all physical and biological stresses can increase its production in plant [2,6,7,13-16]. Therefore, it has many physiologic roles in plant biology and so, its presence in plants and their products is fully logical and expected.

According to accepted standards, the presence of 120-460 mgL-1 (with mean 140mgL-1) methanol in kinds of fresh and canned juices is naturally permitted [17-21]. So, its existence in herbal distillates shows their reality [22]. Herbal distillates are usually colorless liquids and obtained from the condensed steam of different parts of medicinal plants. They usually consist of water as the main factor, different essences, drugs, and organic compounds, and are frequently being used for different purposes in food regimen of some countries. There are many reports about the presence of high levels of methanol in these products. Prolonged drinking of such drinks can lead to chronic type of methanol intoxication. So, measuring and decreasing of methanol in them is recently considered [18-22]. The only reported symptom of this type of intoxication is apparently the blurred vision leading to nervous blindness [22].

Unfortunately, despite the extended usage of herbal distillates in Iran, there is not any applicable standard about methanol allowable concentration in them. This may be due to the lack of necessary standards and access to an inexpensive and simple diagnostic method in this regard [18-20,22]. Direct detection or determination of methanol needs advanced and very expensive apparatus (usually gas chromatography or GC), high technical knowledge or experience. Sometimes, the difficult and troublesome of techniques of sample preparation make their usage impossible in common labs [7,23-28]. Therefore, access to a cheap and sensitive method along with the needed standards for the quality control of the methanol content of the herbal distillates seems to be necessary.

Methanol’s oxidization is lead to formaldehyde formation that can be determined using different chemical methods such as Chromotropic Acid (CA) one [29]. This method has been recommended as a reference method to measure methanol in alcoholic drinks by Association of Official Analytical Chemists (AOAC) [30]. This method has three steps. Methanol firstly oxidizes into formaldehyde and then, formic acid by potassium permanganate in acidic medium. In the next step, the violet color of the excess Manganese (Mn) is faded by sodium hydrogen sulfite through transforming violet Mn7+ to colorless Mn2+ to see the possible positive result. At the final step, the formic acid is reduced to formaldehyde to react with CA in hot concentrated sulfuric acid vicinity that is accompanied by the appearance of the violet complex. The intensity of appeared color depends on the samples methanol level that can be determined at 575nm.

Recently, an Iranian company has produced two qualitative and quantitative diagnostic kits with easy application based on this method to determine and measure the methanol content of herbal distillates that the efficacy of them has been proven in previous studies [18-20,22]. Therefore, the formulation of a suitable standard regarding the permitted dose of methanol in these products can be effective for their control at this time. So, the aim of the current study was the quantification of methanol in some kinds of herbal distillates by two chemical and GC methods to introduce the factors having an important role in the standardization of their methanol content.

Materials and Methods

In this study, the methanol contents of 60 different samples (ten kinds of herbal distillates from six diverse companies) were examined by both chemical (kit) and GC methods, and finally, the obtained results were used to investigate the principles must be considered for their standardization.


A Gas Chromatography (GC) device (YL 6100 GC model, South Korea) was applied to determine of methanol. The GC system equipped with a Flame Ionization Detector (FID) and TR-25, capillary column (30m×0.53mm×1.5μm). A 10μL Hamilton syringe (made by Australia) was used to inject samples. Also, a spectrophotometer (6405 UV/VIS Jenway, England) is used for performance of tests by chemical kit.


The needed methanol and ethanol for preparation of standard solutions for GC method was prepared with analytical grade from Merck (Darmstadt, Germany) and used without further purification. A newly designed (by the author) kit that has been produced by an Iranian company (Arya Mabna Tashkhis Co., Tehran, Iran) was used in the chemical method of methanol measuring. This kit contains five reactants (shown by A, B, C, D and E), five methanol standards with 0, 12.5, 25, 50 and 100 mgL-1 concentrations and an instruction brochure available in the pack. Also, the 60 different industrial herbal distillates (Mentha spicata L., Mentha pulegium L., Urtica dioica L., Trigonella foenum-graecum L., Cichorium intybus L., Alhagi maurorum L., Anethum graveolens L., Rosa damascena L., Citrus aurantium L., and Salix aegyptiaca L.) were prepared from different local commercial stores (Rasht, Gilan province, Iran). Their producing and expiring dates were nearly close together (maximum one-month) whereas, the difference was nearly up to five months in products of all different companies. These samples were known selected based on some specific characteristics (such as origin tissue, amount and circumstances of using, etc.) to better interpret the gained results and the needed basics are discussed more easily. Also, deionized double Distillated Water (DW) was used for preparatin of all standards solutions and dilution of the samples.

Preparation of samples and Standard solutions

Five standard solutions containing 0, 12.5, 25, 50 and 100 mgL-1 of methanol with 100mgL-1 ethanol (as the internal standard) was prepared by a serial method to use in GC method. For determination of samples’ methanol by GC, some ethanol solution was added into 10mL of each sample to obtain 100mgL-1 concentration of it. Whereas, for chemical method, one volume of each sample (triplicate) was diluted with four volume of DW (1:4 ratio) to obtain 1:5 dilution ratio to examine as double.

The kit procedure

According the kit brochure, 0.2mL of each standard and all diluted samples were poured into separated previously labeled test tubes with 50μL of reactants A and B (sulfuric acid and potassium permanganate solutions, respectively) and well shaken. After five minutes, reactant C (sodium hydrogen sulfite) was added and the mixture was shaken hardly until became colorless. 50μL of reactant D (CA solution) and one mL of reactant E (concentrated sulfuric acid) were then added to the tubes and shaken. After spontaneously cooling down at room temperature, their absorbance was read at 575nm in contrast of DW blank. Finally, the methanol content of samples was computed in comparison to the standard curve by multiplication of the result into the dilution factor (five).

GC procedure

Helium carrier gas (at a linear 6ml min-1 rate) was used for methanol separation. All standards and samples were directly injected (2μL with 1:20 split ratio) to GC apparatus as triplicate at 80°C column oven temperature (isothermal condition). The injector port and detector temperatures were 200 and 300°C, respectively. Then, the obtained results were corrected based on internal standard peak and finally, the average of three replicate results were used as last results for next calculations.


Methanol content of all samples was determined using both GC and chemical methods. The results are shown in Table 1. According to the kit manufacturer’s claim (in the brochure), the kit limit of quantification (LOQ) is 7mgL-1 in herbal distillates. As it is seen in Table 1, all samples had methanol more than the mentioned LOQ.

Citation: Rafizadeh A, Shariati Sh, Safarzadeh Vishekaei MN, Honarchian Masihi P and Rafizadeh M. Investigating the Factors Having the Important Role in the Standardization of Herbal Distillates’ Methanol Content. Austin Environ Sci. 2021; 6(2): 1056.