Phosphodiesterase type 5 inhibitors (PDE-5i) are the first-line medication for oral erectile dysfunction, which are used according to the prescription of doctors. However, these substances have been found illegally in supplementary foods. The quality and safety of dietary supplements for enhancing male sexual performance have been questioned, raising the need for continual development of analytical methods. Liquid chromatography coupled with high-resolution mass spectrometry has become one of the most effective methods to identify and measure PDE-5i concentration. In this research, we focused on (i) developing and validating an effective screening and quantitation method for more than 53 PDE-5i in ingredients and supplementary products using LC-Q-Exactive after a simple sample extraction and (ii) assessing PDE-5i content in natural-based supplementary products available in Vietnam market. The extraction method used a small amount of organic solvent, which makes it more environmentally friendly (greener). The developed method has a limit of detection of 0.4 mg/kg, a limit of quantitation of 1.2 mg/kg, recoveries from 80 to 110%, and repeatability lower than 15%. Ninety-two herbal supplementary foods and ingredients used for enhancement of male sexual performance available in Vietnamese markets were collected. Fourteen PDE-5i including conventional and novel analogous were detected and measured in eighteen food supplements and two formulation ingredient samples.
Phosphodiesterase type 5 (PDE-5) is an enzyme responsible for the breakdown of cyclic guanosine monophosphate (cGMP) in the corporal smooth muscle [
PDE-5i, namely sildenafil, tadalafil, vardenafil, and their analogs, have been added in supplementary foods, which were supposed to be made of natural ingredients. The presence of PDE-5i in these supplement foods without labeling is consumer deception. In 2013, J.H. Lee et al. reported more than 46 PDE-5i analogs in various forms of health food products in the online and offline market of Korea [
The rapid and accurate identification and measurement of popular and unknown PDE-5i have been improved by numerous analytical techniques including high-performance liquid chromatography (HPLC) [
The development of high-resolution mass spectrometry (HRMS) techniques has made screening applications more selective than conventional MS techniques. Currently, time-of-flight mass spectrometry (TOF) or Orbitrap mass spectrometry techniques can achieve high mass accuracy (below 5 ppm). In particular, Orbitrap mass spectrometry can perform high-resolution MS/MS allows both the screening of unknown compounds and the quantification of target substances. Therefore, liquid chromatography coupled with HRMS has become one of the most effective methods to identify and measure PDE-5i concentration. Our goals are (i) developing and validating a screening and quantitation method for PDE-5i in supplementary products and (ii) assessing PDE-5i content in natural-based supplementary products and ingredients available in the Vietnam market.
Fifty-three PDE-5i standards were obtained from Toronto Research Chemicals (Martin Ross Avenue, North York, Ontario, Canada) and LGC Standards (GmbH Mercatorstrasse, Wesel, Germany). Methanol, acetonitrile, n-hexane, and other organic solvents were purchased from Merck (Darmstadt, Germany). Formic acid and ammonium formate were purchased from Sigma-Aldrich (St. Louis, USA). Deionized water (18.2 MegaOhm.cm) was purified using a Milli-Q system (Millipore, Co., Bedford, MA, USA).
Each solid standard was accurately weighed about 10 mg and diluted by 10 mL methanol to obtain a 1000
Ninety-two supplement foods and ingredients specific for the enhancement of male sexual performance were collected in local stories in Vietnam.
Samples in the form of tablets were crushed into a fine powder. In samples in the form of hard-shelled capsules or soft-gel capsules, the capsules had been removed and the inside content only has been homogenized. Each homogeneous sample was weighed 0.10 g in a 15 mL centrifuge tube by an analytical balance. Then, 4.0 mL of acetonitrile: water (1 : 1, v/v) was added and mixed well before being sonicated for 30 minutes. The extract was centrifuged for 5 minutes at a speed of 6000 rpm. The solution was separated from the residue and filtered through a polytetrafluoroethylene filter (0.2
The sample solutions were analyzed by the UltiMate 3000 UHPLC system coupled with Q-exactive (Thermo Fisher Scientific Inc., USA). The separation was conducted on Waters BEH C18 (100 mm × 1.7
The Q-exactive was equipped with heated electrospray ionization (HESI) source with the following parameters: HESI temperature of 320°C, the capillary temperature of 350°C, spray voltage of 5000 V, sheath gas flow of 30 arbitrary units, the auxiliary gas flow of 10 arbitrary units. The mass spectrometer was operated in the full MS/data-dependent MS/MS mode (full MS-dd-MS/MS) with the following parameters: scan range 200–2000 m/z, resolution 70,000 FWHM (defined for m/z 200; 3 Hz), automatic gain control (AGC) target 1
Mass spectrometric parameters for identification and retention time of PDE-5i.
No | PDE-5i | Chemical formula | Retention time (min) | Ion type | Precursor ion (m/z) | Product ions (m/z) |
---|---|---|---|---|---|---|
1 | Mirodenafil | C26H37N5O5S | 7.57 | M + H | 532.2588 | 99.09167; 296.13935; 312.13427; 70.06513; 56.04948; 84.0682; 210.06619; 129.10224; 88.07569; 121.03964 |
2 | Noracetildenafil | C24H32N6O3 | 13.96 | M + H | 453.2609 | 70.06513; 97.07602; 113.10732; 98.08385; 58.06513; 56.04948; 297.1346; 166.09749; 325.12952; 353.16082 |
3 | Desmethyl fondenafil | C23H30N6O3 | 15.29 | M + H | 439.2459 | 339.14505; 311.11395; 396.20255; 374.42169; 319.71493; 196.38647; 165.01724; 120.37976; 99.0919; 73.11301 |
4 | N-Octylnortadalafil | C29H33N3O4 | 15.64 | M + H | 489.3126 | 169.07602; 135.04406; 204.08078; 262.08626; 338.22269; 115.05423; 197.07094; 264.10191; 130.06513; 232.07569 |
5 | Acetylvardenafil | C25H34N6O3 | 16.01 | M + H | 467.2765 | 169.09715; 70.06513; 84.08078; 341.16082; 111.09167; 72.08078; 127.12297; 97.07602; 112.0995; 110.06004 |
6 | Lodenafil carbonate | C47H62N12O11S2 | 16.07 | M + H | 1048.642 | 112.0995; 82.06513; 58.06513; 97.07602; 111.09167; 56.04948; 487.2122; 83.06037; 84.08078; 283.11895 |
7 | Hydroxyacetildenafil | C25H34N6O4 | 16.13 | M + H | 483.2714 | 97.07602; 70.06513; 127.08659; 143.11789; 100.07569; 297.1346; 88.07569; 166.09749; 112.0995; 128.09441 |
8 | Carbodenafil | C24H32N6O3 | 16.23 | M + H | 453.2609 | 311.11387; 339.14517; 166.09749; 255.12404; 69.04472; 97.07602; 225.07709; 70.06513 |
9 | Acetildenafil | C25H34N6O3 | 16.48 | M + H | 467.2765 | 111.09167; 97.07602; 70.06513; 84.08078; 72.08078; 127.12297; 112.0995; 297.1346; 56.04948; 166.09749 |
10 | Descarbonsildenafil | C21H30N6O4S | 16.59 | M + H | 463.2129 | 418.15475; 311.15069; 432.17177; 406.15494; 361.13279; 344.14795; 283.11908; 238.83536; 192.99106; 175.69979; 151.05383; 125.02768; 87.09227; 72.08158; 58.066 |
11 | Piperiacetildenafil | C24H31N5O3 | 16.75 | M + H | 438.25 | 98.09643; 70.06513; 297.1346; 55.05423; 166.09749; 341.16082; 69.04472; 325.12952; 86.09643 |
12 | Dimethylacetildenafil | C25H34N6O3 | 16.96 | M + H | 467.2765 | 84.08078; 127.12297; 112.0995; 111.09167; 70.06513; 297.1346; 58.06513; 166.09749; 325.1659; 410.21867 |
13 | Hydroxyvardenafil | C23H32N6O5S | 17.09 | M + H | 505.2228 | 169.09715; 344.14791; 99.09167; 110.06004; 299.11387; 123.09167; 58.06513; 56.04948; 68.01309; 82.06513 |
14 | N-Desethylvardenafil | C21H28N6O4S | 17.10 | M + H | 461.1966 | 169.09715; 344.14791; 110.06004; 299.11387; 316.11661; 123.09167; 68.01309; 82.06513; 56.04948 |
15 | Piperazonifil | C25H34N6O4 | 17.10 | M + H | 483.2726 | 465.26167; 436.22269; 429.52412; 408.22737; 380.20885; 339.1819; 297.13488; 266.45598; 244.12189; 203.11833; 153.1027; 127.08668; 99.0923; 72.08144 |
16 | Vardenafil | C23H32N6O4S | 17.20 | M + H | 489.2279 | 169.09715; 344.14791; 110.06004; 299.11387; 72.08078; 123.09167; 70.06513; 376.1074; 68.01309; 113.10732 |
17 | Avanafil | C23H26ClN7O3 | 17.33 | M + H | 484.1858 | 155.02582; 375.12184; 105.03349; 77.03858; 95.04914; 53.03858; 357.11128; 233.1033; 67.05423; 221.1033 |
18 | Isosildenafil | C22H30N6O4S | 17.35 | M + H | 475.2122 | 58.06513; 99.09167; 283.11895; 100.0995; 56.04948; 253.072; 70.06513; 311.15025; 225.07709 |
19 | Hydroxyhomosildenafil | C23H32N6O5S | 17.36 | M + H | 505.2228 | 99.09167; 70.06513; 58.06513; 84.0682; 97.07602; 283.11895; 88.07569; 129.10224; 112.0995; 311.15025 |
20 | N-Desmethylsildenafil | C21H28N6O4S | 17.37 | M + H | 461.1966 | 85.07602; 283.11895; 311.15025; 56.04948; 299.10868; 225.07709; 254.07983; 253.072; 377.1278; 344.14791 |
21 | Sildenafil | C22H30N6O4S | 17.39 | M + H | 475.2122 | 58.06513; 100.0995; 99.09167; 56.04948; 283.11895; 70.06513; 311.15025; 225.07709; 299.11387 |
22 | Homosildenafil | C23H32N6O4S | 17.47 | M + H | 489.2279 | 72.08078; 58.06513; 99.09167; 113.10732; 70.06513; 283.11895; 84.08078; 71.07295; 114.11515; 311.15025 |
23 | Acetaminotadalafil | C23H20N4O5 | 17.60 | M + H | 433.1507 | 204.08078; 262.08626; 135.04406; 205.0886; 233.08352; 232.07569; 169.07602; 191.07295; 263.09408; 250.08626 |
24 | Aminotadalafil | C21H18N4O4 | 17.60 | M + H | 391.1401 | 204.08078; 135.04406; 262.08626; 233.08352; 169.07602; 232.07569; 250.08626; 191.07295; 203.07295 |
25 | Sildenafil N-oxide | C22H30N6O5S | 17.60 | M + H | 491.2071 | 99.09167; 56.04948; 70.06513; 404.1387; 344.14791; 58.06513; 97.07602; 283.11895; 311.15025; 377.1278 |
26 | Cyclopentylnafil | C26H36N6O4S | 17.70 | M + H | 529.2592 | 461.19682; 377.13029; 344.1461; 313.16608; 277.28223; 237.59493; 210.18739; 169.09731; 142.733; 98.09704; 75.59057 |
27 | Dimethylsildenafil | C23H32N6O4S | 17.71 | M + H | 489.2279 | 99.09167; 71.07295; 56.04948; 113.10732; 70.06513; 283.11895; 311.15025; 84.08078; 377.1278; 225.07709 |
28 | Nortadalafil | C21H17N3O4 | 17.77 | M + H | 376.1292 | 204.08078; 262.08626; 135.04406; 233.08352; 232.07569; 169.07602; 191.07295; 254.0924; 250.08626 |
29 | Udenafil | C25H36N6O4S | 17.98 | M + H | 517.2592 | 84.08078; 112.11208; 283.11895; 58.06513; 325.1659; 299.11387; 81.06988; 255.12404; 79.05423; 82.06513 |
30 | Benzamidenafil | C19H23N3O6 | 18.02 | M + H | 390.166 | 151.07536; 107.04914; 135.04406; 91.05423; 79.05423; 105.03349; 90.0464; 136.05188; 65.03858; 93.03349 |
31 | Norneovardenafil | C18H20N4O4 | 18.07 | M + H | 357.1557 | 169.07602; 110.06004; 329.12443; 328.11661; 123.09167; 68.01309; 300.08531; 55.05423; 82.06513; 95.06037 |
32 | Propoxyphenyl-homohydroxysildenafil | C24H34N6O5S | 18.13 | M + H | 519.2384 | 99.09167; 70.06513; 283.11895; 84.0682; 97.07602; 299.11387; 129.10224; 88.07569; 112.0995; 255.12404 |
33 | O-desethyl-o-propyl sildenafil | C23H32N6O4S | 18.14 | M + H | 489.2285 | 447.1196; 416.97849; 391.14451; 347.08125; 325.16612; 283.11909; 252.20022; 230.78935; 193.95794; 163.05384; 107.28071; 100.10004; 91.75806; 70.06595; 58.06599 |
34 | 2-Hydroxypropyl nortadalafil | C24H23N3O5 | 18.20 | M + H | 434.1711 | 135.04406; 169.07602; 204.08078; 262.08626; 284.13935; 197.07094; 130.06513; 115.05423; 232.07569; 312.13427 |
35 | Propoxyphenyl aildenafil | C24H34N6O4S | 18.39 | M + H | 503.2447 | 461.19514; 391.14362; 347.08197; 325.16609; 283.11925; 256.09442; 189.66799; 159.62481; 137.61562; 113.1077; 99.09223; 91.76256; 71.07376 |
36 | Acetil acid | C18H20N4O4 | 18.45 | M + H | 357.1557 | 285.1345; 300.08487; 313.16528; 273.23201; 234.78161; 329.12441; 57.55269; 76.77712; 91.7671; 128.3394; 166.09776 |
37 | Tadalafil | C22H19N3O4 | 18.69 | M + H | 390.1448 | 204.08078; 135.04406; 262.08626; 169.07602; 205.0886; 232.07569; 233.08352; 240.11314; 268.10805; 250.08626 |
38 | Depiperazino-thiosildenafil | C17H20N4O4S2 | 18.80 | M + H | 409.1012 | 381.06896; 365.03707; 352.03045; 328.13556; 300.10448; 272.07283; 253.43238; 218.38203; 200.90222; 182.07341; 146.98571; 130.30821; 91.75983; 69.50264 |
39 | Mutaprodenafil | C27H35N9O5S2 | 18.95 | M + H | 630.2282 | 142.00711; 602.23247; 560.22363; 516.1504; 489.22772; 439.15549; 404.13937; 377.12856; 344.14797; 312.15851; 288.21153; 219.20777; 163.22714; 113.10774; 84.98616 |
40 | Gendenafil | C19H22N4O3 | 19.24 | M + H | 355.1765 | 327.14517; 285.1346; 298.10604; 256.09548; 311.11387; 69.04472; 120.04439; 154.0611; 313.1659; 166.09749 |
41 | Hydroxychlorodenafil | C19H23ClN4O3 | 19.26 | M + H | 391.1531 | 313.12952; 285.1346; 363.12184; 256.09548; 120.04439; 69.04472; 166.09883; 78.99452; 327.14517; 255.08765 |
42 | Hydroxythiovardenafil | C23H32N6O4S2 | 19.57 | M + H | 521.1999 | 167.06375; 360.12506; 99.09167; 315.09037; 138.02462; 150.10257; 58.06513; 299.09611; 70.06513; 332.09307 |
43 | Chloropretadalafil | C22H19ClN2O5 | 19.65 | M + H | 427.1055 | 135.04406; 274.08559; 204.08078; 216.08078; 189.06988; 262.08674; 244.0735; 302.08117 |
44 | Chlorodenafil | C19H21ClN4O3 | 19.79 | M + H | 389.1375 | 361.10619; 285.1346; 311.11387; 154.0611; 166.09749; 69.0573; 256.09548; 76.97887; 165.0183 |
45 | Benzylsildenafil | C28H34N6O4S | 20.01 | M + H | 551.2435 | 91.05423; 65.03858; 134.09643; 377.1278 |
46 | Nitrodenafil | C17H19N5O4 | 20.09 | M + H | 358.151 | 330.11968; 316.11661; 154.0611; 256.09548; 68.0369; 255.08765; 313.11694; 227.09274; 269.1033; 136.05054 |
47 | Pseudovardenafil | C22H29N5O4S | 20.21 | M + H | 460.2013 | 169.09715; 110.06004; 344.14791; 299.11387; 123.09167; 284.12678; 68.01309; 82.06513; 55.05423; 95.06037 |
48 | Imidazosagatriazinone | C17H20N4O2 | 20.32 | M + H | 313.1659 | 285.1346; 256.09548; 120.04439; 68.0369; 255.08765; 241.072; 269.1033; 69.04472; 154.0611; 94.02874 |
49 | Propoxyphenylthio-hydroxyhomosildenafil | C24H34N6O4S2 | 20.55 | M + H | 535.2156 | 99.09167; 70.06513; 56.04948; 299.09611; 58.06513; 84.0682; 129.10224; 315.09037; 88.07569; 271.10119 |
50 | Thiohomosildenafil | C23H32N6O3S2 | 20.55 | M + H | 505.205 | 72.08078; 99.09167; 113.10732; 56.04948; 299.09611; 70.06513; 84.08078; 327.12741; 71.07295; 355.15806 |
51 | Hydroxythio-homosildenafil | C23H32N6O4S2 | 20.56 | M + H | 521.1999 | 99.09167; 70.06513; 58.06513; 84.0682; 299.09611; 129.10224; 97.07602; 88.07569; 327.12741; 112.0995 |
52 | Norneosildenafil | C22H29N5O4S | 20.64 | M + H | 460.2013 | 283.11895; 84.08078; 299.09611; 311.15025; 154.0611; 316.11661; 255.12404; 344.14791; 166.09749 |
53 | Thiosildenafil | C23H32N6O3S2 | 21.47 | M + H | 505.205 | 99.09167; 71.07295; 299.09611; 113.10732; 56.04948; 70.06513; 327.12741; 84.08078; 241.0542; 298.08828 |
Real samples were first screened PDE-5i as the scheme in Figure
Screening workflow for unknown substances.
First of all, parameters for detecting PDE-5i in Q-exactive mass spectrometer should be set up before further optimization. All PDE-5i have chemical structures suitable for being ionized by electrospray ionization source in positive mode. A 500
PDE-5i are less polar compounds, so they can be analyzed by the C18 base chromatography column. Because of their similar structures, PDE-5i should be separated by a chromatography column that has a small particle size. Therefore, we chose BEH C18 (100 mm × 1.7
Referring to previous studies [
Comparison of the intensity of four representative PDE-5i with different extraction solvents. (a) Acetildenafil. (b) Acetaminotadalafil. (c) Hydroxyvardenafil. (d) Avanafil.
For the soft-gel capsule sample, however, we added a second solvent to remove oily components of samples before injecting the extraction into the LC-HRMS system. Three organic solvents including n-hexane, diethyl ether, and ethyl acetate were examined, and the result was shown in Figure
Comparison of the intensity of four representative PDE-5i after cleaning with three different solvents. (a) Acetildenafil. (b) Acetaminotadalafil. (c) Hydroxyvardenafil. (d) Avanafil.
The developed method had been validated before applied to real samples analysis. The specificity of the method was proved by mass accuracy of precursor ions and productions and comparison between blank samples and standard materials. The validation parameters were showed in Table
All the compounds showed ME less than 10%. Thus, calibration curves in the standard solution can be used to calculate the concentration of PED-5i in real samples.
For screening purposes, 92 real samples were analyzed by the developed method (
Result of screening PDE-5i in 92 samples.
PDE-5i | Number of detected samples |
---|---|
Nortadalafil | 11 |
Tadalafil | 10 |
Sildenafil | 9 |
Hydroxyhomosildenafil | 6 |
Hydroxythiohomosildenafil | 6 |
Homosildenafil | 6 |
Thiohomosildenafil | 1 |
Sildenafil N-oxyde | 2 |
Chloropredadalafil | 1 |
Propoxyphenyl-homohydroxysildenafil | 1 |
Propoxyphenylaildenafil | 1 |
Hydroxythiovardenafil | 1 |
Methisosildenafil | 1 |
N-hydroxyethyl dithio-desethyl carbodenafil | 2 |
After screening, positive samples were confirmed and quantified. The concentration of PDE-5i in these samples was calculated by calibration curves and presented in Table
Concentration of PDE-5i detected in real samples.
Sample | PDE-5i | Concentration (mg/g) |
---|---|---|
S 1 | Nortadalafil | 50.0 ± 0.13 |
Tadalafil | 11.6 ± 0.05 | |
Sildenafil-N-oxide | ∼0.073 | |
S 2 | Nortadalafil | 4.41 ± 0.05 |
Chloropretadalafil | ∼0.006 | |
S 3 | Sildenafil | 22.2 ± 0.11 |
Tadalafil | 0.39 ± 0.007 | |
Sildenafil-N-oxide | ∼0.06 | |
S 4 | Propoxyphenylaildenafil | 1.02 ± 0.06 |
Thiohomosildenafil | 0.78 ± 0.005 | |
Homosildenafil | ∼0.031 | |
Methisosildenafil | ∼0.03 | |
S 5–9 | Hydroxyhomosildenafil | 1.05–20.1 |
Hydroxythiohomosildenafil | 0.78–22.5 | |
S 10 | Tadalafil | ∼0.08 |
S 11 | Tadalafil | 10.2 ± 0.05 |
Chloropretadalafil | 5.25 ± 0.06 | |
S 12 | Nortadalafil | 12.2 ± 0.05 |
Chloropretadalafil | 0.25 ± 0.06 | |
S 13 | Nortadalafil | 10.2 ± 0.04 |
Chloropretadalafil | 5.25 ± 0.10 | |
S 14 | Acetil acid | 0.13 ± 0.06 |
S 15 | Tadalafil | 0.12 ± 0.05 |
S 16 | Sildenafil | 5.25 ± 0.11 |
Tadalafil | 4.77 ± 0.15 | |
Aminotadalafil | 0.52 ± 0.05 | |
Sildenafil N-oxyde | 0.56 ± 0.06 | |
S 17 | Nortadalafil | 0.52 ± 0.06 |
Chloropretadalafil | 4.77 ± 0.10 | |
S 18 | Nortadalafil | 12.1 ± 0.06 |
M 1 | Nortadalafil | 170.0 ± 1.22 |
M 2 | Hydroxythiohomosildenafil | 226.8 ± 1.36 |
Hydroxythiovardenafil | 216.8 ± 2.00 | |
Propoxyphenyl-homohydroxysildenafil | 0.13 ± 0.06 | |
Hydroxyhomo-sildenafil | ∼0.048 | |
Hydroxyvardenafil | ∼0.047 |
S: supplementary food, M: medicine ingredient, “S 5–9” indicates samples: S 5, S 6, S 7, S 8, S 9.
In this study, we have developed and validated a rapid screening and quantitation method using LC-HRMS for more than 53 PDE-5i in ingredients and supplementary products for enhancing male sexual performance. The validation parameters of this method, such as LODs, LOQs, recoveries, and regression coefficients, were acceptable according to the requirement of AOAC for an analytical method. The success of this method demonstrated the utilization of the fragmentation mass spectra library for analytes confirmation. The developed method was applied to analyze 92 natural-based ingredients and supplementary products available in the Vietnam market. We had screened and detected 14 PDE-5i. The results of real samples analysis implied that the manufacturers had deceived customers by not declaring PDE-5i on the label but adding these ingredients in the products. Thus, our study provides a warning on the quality control of supplementary foods to avoid any health risks to the community.
The data used to support the findings of this study are available within the article, the support information in word form, and from the corresponding author upon request.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
This work has received funding from the National Institute of Food Control, Vietnam (project number: NIFC.DTCS.20.06).
Supplementary information is provided in word form.