Detection Of 15 Afrodyn Drugs Illegally Added in Sex-enhancing Product By HPLC-MS/MS With Solid Nuclear Particle Chromatographic Column

Abstract:

Objective To develop an HPLC-MS/MS method with solid nuclear particle chromatographic column to determine 15 afrodyn drugs illegally added in sex-enhancing product. Methods The analysis was performed by an HPLC-MS/MS system, with Cortecs-C18 (150 mm × 4.6 mm, 2.7 μm) column. Multiple-reaction monitoring (MRM) was performed to identify and quantify phentolamine mesylate, noracetildenafil, acetildenafil, vardenafil, sildenafil, hydroxyhomosildenafil, homosildenafil, aminotadalafil, tadalafil, thioaildenafil, pseudovardenafil, norneosildenafil, methyltestosterone, stanozolol and danazol, which were extracted with methanol by ultrasonic. Results The linear calibration curves of 15 chemical components mentioned above were obtained at r 2 ≥ 0.995 4 with better separation degree and wide linear range. The precision of the method was shown by RSD (n = 6) from six estimated values ranged from 0.9% to 3.3%. The recovery rates determined at three adding concentrations were from 88.3% to 109.5% and the RSD was from 0.9% to 5.2%. The range of limit of quantitation (LOQ) were from 0.015 μg/mL to 0.30 μg/mL and The RSD of inter-day and intra-day precision were from 0.9% to 4.2% and from 1.0% to 4.5%, respectively; Twenty batches of positive samples of 25 batches of sex-enhancing products were detected to be added with sildenafil and with an unqualified rate of 80%. Conclusion The method is specific and simple to detect that 15 afrodyn drugs were added illegally in sex-enhancing product. At the same time, the high positive detection rate should attract the attention of the department concerned, and the corresponding policies should be issued to guarantee the health and safety of the people.

 

Keywords: solid nuclear particle chromatographic column; HPLC-MS/MS; sex-enhancing product; afrodyn; added illegally; phentolamine mesylate; noracetildenafil; acetildenafil; vardenafil; sildenafil; hydroxyhomosildenafil; homosildenafil; aminotadalafil; tadalafil; thioaildenafil; pseudovardenafil; norneosildenafil; methyltestosterone; stanozolol; danazol

 

100% Natural Herbal Cistanche Supplements For Improve Sexual Functions In long Term Treatment

 

 

In recent years, some criminals have used chemical drugs added to health foods and other products to impersonate drugs, and mislead and deceive consumers by promoting the functions and medicinal effects of the products. This type of case is also known as the so-called non-drug impersonation of drug cases. In recent years, the products most involved in such cases are so-called "sexual health products".
These sexual health products are emerging in the market in an endless stream, mainly sold online and by phone. Their characteristics are that their packaging and product names have obvious sexual connotations, such as "American Viagra", "Plant Viagra", "7 Days of War", etc.
These products are claimed to have no side effects and strong effects, but in fact, they illegally add aphrodisiac chemical drugs. Taking sexual health products that illegally add these drug ingredients can cause headaches, dizziness, blurred vision, and cover up the occurrence of cardiovascular diseases. In severe cases, it can lead to high blood pressure or sudden death, which seriously endangers human health [1-2]. In recent years, there have been many reports discussing the determination of illegally added chemical ingredients in health foods and Chinese patent medicines [2-17]. Solid core particle chromatography technology is a new chromatographic filling technology developed in recent years. This technology optimizes the operating back pressure of high-performance liquid chromatography, maximizes the separation efficiency of ordinary high-performance liquid chromatography by improving the separation degree and peak capacity, and makes the analysis efficiency comparable to ultra-high performance liquid chromatography, and is more versatile and popular. In this experiment, solid core particle chromatography technology was applied to HPLC-MS/MS to simultaneously and rapidly detect 15 aphrodisiac chemical components in sexual health care.

 

1 Materials and methods

1.1 Instruments

High performance liquid chromatography-tandem quadrupole mass spectrometry, equipped with Agilent 1260 high performance liquid chromatograph, Agilent 6410 tandem quadrupole mass spectrometer, Agilent, USA; Mili-Q deionized water generator, Millipore, USA.

1.2 Samples and reagents
A total of 25 batches of sexual health care products were measured in the experiment, all of which were sampled by Hangzhou Market Supervision and Administration Bureau. Reference substances phentolamine mesylate (batch number 100110-200602, mass fraction 99.9%), methyltestosterone (batch number 100172-200503, mass fraction 100%), stanozolol (batch number 100209-201003, mass fraction 99.8%), and danazol (batch number 100126-200402, mass fraction 99.3%) were purchased from the China Food and Drug Inspection Institute. Redenafil (batch number 1591-080A3, mass fraction 99.2%), redenafil (batch number 1218-018A12, mass fraction 99.8%), vardenafil (batch number 1132-035A1, mass fraction 99.7%), sildenafil (batch number 1358-057A1, mass fraction 99.3%) were purchased from the China Food and Drug Inspection Institute. 100%), hydroxyhomosildenafil (batch number 1199-001A1, mass fraction 98.5%),homosildenafil (batch number 1220-030A3, mass fraction 99.2%), aminotadalafil (batch number 1638-004A2, mass fraction 99.7%), tadalafil (batch number 1156-051A1, mass fraction 99.7%), thio-edenafil (batch number 1214-006A3, mass fraction 99.5%), pseudovardenafil (batch number 1217-011A2, mass fraction 99.5%), and narmosildenafil (batch number 1646-055A2, mass fraction 99.9%) were purchased from TLC Pharmaceutical Standards, Canada. Formic acid was analytical grade and methanol was chromatographic grade.

 

1.3 Chromatographic conditions

The chromatographic column is CORTECS-C18 (150 mm×4.6 mm, 2.7 μm); the mobile phase is 0.1% formic acid aqueous solution-methanol, and the gradient elution program is: 0-15 min, 50%-90% methanol; 15-20 min, 90%-100% methanol; 20-25 min, 100%-50% methanol;
column temperature 40 ℃; volume flow rate 0.3 mL/min; injection volume 10 μL.

 

1.4 Mass spectrometry conditions

Ion source: electrospray ion source (ESI), positive ion detection mode (ESI+); multiple reaction monitoring (MRM mode); scanning with segmented MRM mode; drying gas temperature 350 ℃; drying gas volume flow rate 10 L/min; nebulizer pressure 200 kPa; capillary voltage 4 kV; calibration method: mass axis automatic tuning correction; other mass spectrometry analysis parameters of 15 aphrodisiac chemical components are detailed in Table 1.

 

1.5 Preparation of reference solution

Precisely weigh about 10 mg of each of the 15 reference substances, place them in a 25 mL volumetric flask, and add methanol to prepare reference stock solutions of about 0.4 mg/mL each. Precisely pipette 0.025, 0.05, 0.1, 0.25, and 0.5 mL of the above reference stock solutions into a 10 mL volumetric flask, dilute with methanol to make a series of reference solutions.

1.6 Preparation of test solution
Take 1 g of this product, place it in a 50 mL volumetric flask, add about 25 mL of methanol, ultrasonicate for 30 min (100 W, 56 kHz), cool, and dilute to scale with mobile phase. Filter with a 0.2 μm filter membrane. Since the mass concentrations of the components detected in the positive samples (sexual health products with aphrodisiac chemical components) 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.

100% Natural Herbal Cistanche Supplements For Improve Sexual Functions In long Term Treatment

2 Results

2.1 Chromatographic results

Take 15 prepared reference substance mixed solutions, inject 5 μL, and separate them using solid core particle chromatographic column CORTECS-C18 (150 mm×4.6 mm, 2.7 μm). The results are shown in Figure 1. It can be seen that the three chromatographic peaks of sildenafil, hydroxyhomosildenafil and homosildenafil have similar chemical properties and basically the same retention time. At the same time, the retention time of pseudovardenafil and narmosildenafil is also basically the same. This experiment uses a single standard with the same mass concentration as the mixed reference substance solution to verify. The results show that several components with similar retention times do not have competitive inhibition effects on each other.

Table 1 MS parameters for 15 afrodyn drugs

 

Component	Measured m/z (m/z)	Ionization Energy / V	Ionization Potential (eV)
α-Pinene	120	120	9.39
Limonene	120	120	9.26
β-Caryophyllene	130	130	9.56
Myrcene	100	100	9.12
D-Limonene	100	100	9.22
Terpinolene	120	120	9.34
α-Humulene	130	130	9.47
γ-Terpinene	120	120	9.20
β-Pinene	130	130	9.45
α-Terpineol	120	120	9.39

 

2.2 Linear relationship investigation, detection limit and quantitative limit

Take 5 μL of the prepared reference substance series solutions, inject 5 μL respectively, and use the peak area of ​​each component as the ordinate (Y) and the mass concentration of the reference substance as the abscissa (X) to investigate the linear relationship. The results are shown in Table 2.
Take 1 g of blank sample (Sunshine Man Fire Power Capsule, Nars Biological Co., Ltd., Dali City, Yunnan Province, the test result is negative), add a properly diluted mixed reference solution, prepare the test solution according to the method of "1.6", and obtain the detection limit and quantification limit of 15 kinds of illegal aphrodisiac ingredients when the signal-to-noise ratio is 3 and 10, respectively. The results are shown in Table 2.

100% Natural Herbal Cistanche Supplements For Improve Sexual Functions In long Term Treatment

 

2.3 Method stability test

Weigh 1 g of blank sample (Sunshine Man Fire Power Capsule, Nars Biological Co., Ltd., Dali City, Yunnan Province, the test result is negative), add an appropriate amount of mixed reference solution, and make the mass concentration of the test solution 8 μg/mL. The RSD of the peak area of ​​5 repeated injections within 1 day is taken as the intra-day precision, and the RSD of the peak area of ​​3-day repeated injections (3 repeated injections per day) is taken as the inter-day precision to investigate the stability of the method. The results are shown in Table 2.