Determination Of Sex-enhancing Drugs Illegally Added in Health Care Products By TLC-NIＲS Combined Technology

Abstract: To establish the detection model of sex-enhancing drugs illegally added by the thin layer chromatography ( TLC) and near-infrared spectroscopy ( NIＲS) combined technology,for the rapid qualitative and quantitative analysis of drugs illegally added in health care products． With the content of chemical drugs in health care products is determined by the HPLC method as a reference value． TLC was used in the study． Trichloromethane-methyl alcohol-ammonium hydroxide ( 10 ∶ 1 ∶ 0． 1) was used as the developing solvent and the plate was detected under an ultraviolet lamp． The NIＲ spectrum of chemical component spots on TLC was collected by NIＲS． The rapid qualitative and quantitative model of chemical drugs was established by using the partial least squares regression analysis ( PLS) combined with cross validation method． And validated for model． Established the HPLC analysis method with better separation degree and wide linear range． Established the TLC-NIＲS analysis models of sildenafil and noracetildenafil with wide linear range,and the better stable and predicted results． The correlation coefficient ( Ｒ2 ) ,the root mean square error of calibration validation ( ＲMSECV) and the root mean square error of prediction ( ＲMSEP) were 0． 984,0． 085 and 0． 081; 0． 982,0． 098 and 0． 087, respectively． Using the TLC-NIＲS model,9 batches of in 10 batches of sex-enhancing products were detected to be added sildenafil and an unqualified rate of 90% , the average relative deviation of the content of sildenafil and noracetildenafil of predicted were 1． 86%,1． 96%,respectively． The results showed that the TLC-NIＲS analysis models can be used for above chemical components rapid determination in sex-enhancing products． The method with specific and simple． Which provided the ideas for rapid determination of added illegal drugs in health care products．

 

Natural Cistanche Herbal Supplements For Sexual Performance

 

Keywords: health care products; added illegally; TLC-NIＲS; HPLC; sex-enhancing product

 

As the public's awareness of health care increases, the demand for health products is growing, especially health products labeled as traditional Chinese medicine. Health products are a popular term for health foods, which are called dietary supplements abroad. They are not drugs and have the function of regulating human functions. They are used by specific groups of people, but are not intended to treat diseases [1]. Faced with the temptation of high profits, illegal vendors add excessive doses of cheap chemical raw materials with obvious side effects to health products to enhance the efficacy of products. This has greatly disrupted my country's health products market and posed a serious threat to the health of the people. For example, in 2009, the Xinjiang region's auxiliary hypoglycemic health product "Tangzhining Capsule" added excessive doses of the banned chemical drug glibenclamide, causing many deaths. In 2014, the "Xianxian Weight Loss Capsule" in Shanxi Province added banned chemical drugs sibutramine and sildenafil, causing many people to have adverse reactions or even die. In recent years, in the random inspection and evaluation of health products, it was found that illegal manufacturers added excessive doses of sildenafil to sexual health products and traditional Chinese medicine health preparations to deceive consumers, seriously endangering consumers' health [2,3]; similar phenomena such as excessive doses of banned chemical drugs such as phenafil, biguanides, furosemide and phenolphthalein in sexual health, hypoglycemic and weight loss health products have emerged in an endless stream, and the concealment of the addition is becoming increasingly higher [4,5]; such phenomena also often occur abroad [6]. These illegal acts with wide-ranging impact and great harm deserve deep consideration by drug regulatory authorities, and it is particularly important to develop rapid market sampling and detection methods.

Currently, the detection methods used for illegal addition of chemical drugs in traditional Chinese medicines and health products mainly include thin layer chromatography (TLC) [7], high performance liquid chromatography (HPLC) [8], liquid chromatography-mass spectrometry (LC-MS) [9], gas chromatography-mass spectrometry (GC-MS) [10], ion mobility spectrometry (IMS) and Raman spectrometry (RS) [11].
Among the various detection methods reported, LC-MS, GC-MS and IMS have been studied more, but due to the high price of the instruments and the complex operation, they are not easy to promote and are difficult to popularize in urban and mountainous areas with relatively backward economic development. TLC is one of the four traditional methods for drug identification. It is fast, simple, and has good separation effects. It is widely used in the qualitative and quantitative identification of drugs. However, its disadvantage is that it must have corresponding reference substances or reference medicinal materials [12, 13]. Near infrared spectroscopy (NIRS) has been developed in recent years. It collects the vibration and rotation absorption information of the C-H, O-H, S-H, N-H, C = C and C = O groups of the detected substance in the near infrared spectrum for qualitative and quantitative analysis of substances. It is widely used in agriculture, petroleum, chemical industry, tobacco and food. At present, it has been gradually applied to the pharmaceutical and drug supervision industries. It has great development potential in the rapid qualitative and quantitative detection and analysis of drugs [14, 15].

Therefore, this paper uses the existing popular instruments and test methods to create TLC-NIRS combined technology. Taking sexual health products as an example, a universal qualitative and quantitative detection model for illegal addition of prohibited sexual health chemical drugs is established. It can be used efficiently, quickly and simply for screening and detecting the addition of prohibited chemical drugs in market health products, thereby reducing the detection cost.

1 Instruments and test drugs

1.1 Instruments

Fourier transform Tensor 37 NIＲ spectrometer (BRUKER, Germany), OPUS 5.0 analysis software; LC-20AT high performance liquid chromatograph (Shimadzu, Japan), LC Solution chromatography workstation; BS224S electronic analytical balance (Sartorius, Germany); silica gel GF254 thin layer plate (Qingdao Ocean Biochemical Co., Ltd.).

 

1.2 Test drugs

Sildenafil (510068-201401, 99.9%) and narodenafil (520039-201902, 96.5%) were purchased from China Food and Drug Research Institute, and the structures are shown in Figure 1. Methanol was chromatographically pure, water was ultrapure water, and chloroform, phosphoric acid and ammonia were all analytically pure. Health samples were market samples.

 

Fig. 1 Structures of sex-enhancing chemical drugs

 

2 Methods and Results

2. 1 Preparation of Reference Solution and Sample Solution

Preparation of Reference Solution: Take approximately 25 mg of sildenafil and narendenafil respectively, weigh them accurately, place them in a 5 mL volumetric flask, and dissolve them in methanol to prepare a mixed reference stock solution of approximately 5 mg/mL each; then take approximately 5 mg of sildenafil and narendenafil respectively, weigh them accurately, place them in a 5 mL volumetric flask, and dissolve them in methanol to prepare a single identification reference solution of approximately 1 mg/mL each.

Preparation of Sample Solution: Take the dose of this product once, place it in a 5 mL volumetric flask, add methanol and ultrasonicate for 30 min (100 W, 56 kHz), cool it, add methanol to the mark, and filter to obtain the filtrate. When determining the content, accurately measure 1 mL of each sample solution, dissolve it in the mobile phase to 25 mL, and filter it with a 0.45 μm filter membrane. Since the mass concentrations of the components detected in the positive samples (health products with banned chemical drugs added) vary greatly, the sampling volume can be appropriately increased or the sample solution can be appropriately diluted to make its final mass concentration within the linear range.
Preparation of positive sample solution: Since no positive sample was drawn for naringenfil, a blank sample (Huangjingbajitian Capsule, Luoyang Kangti Biology, the test result was negative) was taken once, and an appropriately diluted naringenfil reference solution was added. The positive sample solution was prepared according to the preparation method under the sample solution, so that the mass concentrations of naringenfil in the positive sample solution were approximately 5, 7.5, and 10 mg/mL, respectively, and 4, 3, and 3 copies of each mass concentration were prepared in parallel.

2. 2 Chromatographic and spectral conditions

2. 2. 1 HPLC chromatographic conditions

Phenomenex ODS C18 (250 mm × 4. 6 mm, 5μm) chromatographic column was used; 45% methanol (A) -55% 0. 3% phosphoric acid aqueous solution (B) was used as the mobile phase for elution for 30 min; flow rate was 1 mL/min, detection wavelength was 290 nm; column temperature was 40 ℃; injection volume was 10 μL.

 

2. 2. 2 TLC chromatographic conditions

Silica gel GF254 thin layer chromatography plate was used, and chloroform-methanol ammonia (10: 1: 0. 1) was used as the developing solvent for development and evaporation. After drying in a dryer for 1 hour, the mark was inspected under a 254 nm ultraviolet lamp (see Figure 2).

 

2. 2. 3 NIRS acquisition conditions

The BRUKER NIRS solid fiber probe was used for measurement, with a wave number range of 12,000 to 4,000 cm-1, 64 scans, a resolution of 16 cm-1, a temperature of 10 to 30 °C, and a humidity of 35% ± 5%. A blank silica gel GF254 thin layer plate was used as a reference, and the spectrum was collected after background subtraction. Each chemical component spot was measured in parallel 5 times, and the NIRS information was saved.

 

2.3 Establishment of HPLC content determination method

2.3.1 Investigation of Linear Relationship

2.0 mL of the mixed reference stock solution (sildenafil 5.25 mg/mL; narcolepsy 5.15 mg/mL) was accurately aspirated and diluted to 10 mL with methanol, and then 0.25, 0.1, 0.5, 1.0, 1.5, 2.0, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0, 36.0, 37.0, 38.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 5 mL, dissolve in mobile phase and make up to 5 mL respectively, shake well.
According to the chromatographic conditions under "2.2.1", sample analysis was performed, peak area was determined, and the regression line equation was calculated with the peak area of ​​each component as the ordinate (Y) and the mass concentration of the reference substance as the abscissa (X): sildenafil is Y = 3 × 106X36 606, r = 0.9989, linear range 0.0525 ~ 1.05 μg; naphthol is Y = 1 × 106X-3 184.4, r = 0.9999, linear range 0.0515 ~ 1.03 μg. The results showed that the above reference substances had good linearity within their respective linear ranges.

 

2.3. 2 Precision, stability, repeatability, and recovery test

Take the mixed reference solution under "2.3.1" and the blank sample (Huangjingbajitian capsule, Luoyang Kangti Biological, the test result is negative) and prepare 6 sample solutions containing sildenafil and nardenafil in parallel according to the method under "2.1". According to the chromatographic conditions under "2.2.1", the mixed reference solution was injected 6 times continuously for precision; one of the sample solutions was injected at 0, 3, 6, 9, 12, and 24 hours for stability; 6 sample solutions were prepared in parallel for repeatability and recovery, and each sample was injected once. The peak areas of sildenafil and nardenafil were determined, and the recovery and its RSD value were calculated. Results The average recovery rates of sildenafil and nardenafil were 100. 16%, 100. 49%; the RSD values ​​were all less than 3% (n = 6), indicating that the instrument had good precision, the sample solution was stable within 24 h, and the method had good recovery and repeatability.

 

2.3.3 Content determination

Accurately aspirate each sample solution, inject and analyze according to the chromatographic conditions under "2.2.1", measure 3 times for each batch, record the peak area, and calculate the content. The results show that among the 10 batches of sexual health samples, 9 batches were found to contain the banned chemical drug sildenafil, and the added dosage varied greatly. The detection rate was 90%. The results are shown in Table 1 and Figure 3.

 

Table 1 Determination results of substances illegally added in 10 sex-enhancing products ( n = 3)

 

Sample	Manufacturer	Sample weight	Content (mg/time)
Gold flower extract	West China Agricultural University	1 × 0.88 g	58.79 ± 1.03
Insect repellent	Hong Kong University of Science and Technology	2 × 0.50 g	73.62 ± 0.21
German chamomile	Haiyun Biological	2 × 0.50 g	22.47 ± 0.23
Early morning dew	Hong Kong University of Science and Technology	2 × 0.50 g	8.90 ± 0.03
Wormwood	USA Habitat	2 × 0.50 g	61.83 ± 0.48
A. niger	West China Agricultural University	2 × 0.50 g	45.20 ± 0.04
Gold thread	Hong Kong University of Science and Technology	2 × 0.50 g	Not detected

 

Fig. 3 HPLC of 10 sex-enhancing products

 

2.4 Establishment of TLC-NIRS qualitative model for sexual health chemical drugs

2.4.1 Spectral information collection

According to the conditions under "2.2.3", the NIRs of the reference spots identified after development on the TLC thin layer plate were collected respectively. The spot volumes of the mixed reference solution were 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 μL, and the spot volumes of the sample solution were 2, 5, and 10 μL, respectively. Each reference spot of the mixed reference and sample with different spot volumes was collected 5 times, and the NIRs overlay diagram is shown in Figure 4. In the experiment, the spectra of the mixed reference with different spot volumes were selected as the calibration set, and the spectra of the samples with different spot volumes were selected as the verification set to ensure that the spectral information of the verification set was within the range of the spectral information of the calibration set.

Fig. 4 Original NIＲ spectra of chemical drugs