Journal of Chromatographic Science 2015;53:38– 46 doi:10.1093/chromsci/bmu010 Advance Access publication March 24, 2014
Article
Determination of Phosphodiesterase-5 Inhibitors and Analogs Using High-Performance Liquid Chromatography with Ultraviolet Detection Elisa A. Nickum* and Cheryl L. Flurer U.S. Food and Drug Administration, Forensic Chemistry Center, 6751 Steger Drive, Cincinnati, OH 45237, USA * Author to whom correspondence should be addressed. Email: [email protected] Received 16 July 2013; revised 10 January 2014
A considerable number of erectile dysfunction products, and dietary supplements suspected of containing phosphodiesterase-5 (PDE-5) inhibitors, have been analyzed by the US Food and Drug Administration. Often these samples are found to contain the approved active pharmaceutical ingredients (APIs) such as sildenafil, tadalafil or vardenafil. However, analogs of these APIs have also been identified in many samples and products containing multiple PDE-5 inhibitors have also been found. A single high-performance liquid chromatography with ultraviolet detection method has been developed for the determination of sildenafil, tadalafil, vardenafil and a number of commonly encountered analogs in pharmaceutical dosage forms and dietary supplement products, including tablets, capsules, bulk powders, troches and liquids. This method was designed as an alternative to methods developed for the determination of a single PDE-5 inhibitor. Using this protocol, 14 PDE-5 inhibitor compounds can be separated and determined in a single analysis.
Introduction The class of drugs known as phosphodiesterase-5 (PDE-5) inhibitors includes sildenafil citrate (Viagraw, Pfizer), tadalafil (Cialisw, Eli Lilly) and vardenafil hydrochloride trihydrate (Levitraw, Bayer), and is marketed and approved for use in the USA in the treatment of erectile dysfunction (ED). During the last decade, these oral drugs have been the first-line treatment for ED as they are easily administered, relatively non-invasive, reversible and well tolerated. However, taking these drugs can be dangerous to those who suffer from certain comorbid conditions such as hypertension, heart disease or diabetes, or those who take nitrates, a-blockers or blood-thinning medication (1). As a result, many men seek alternative medicines, in the form of herbal and dietary supplements, as a therapy for ED with the belief that these products are safe. Studies indicate that these products are often found to contain the active pharmaceutical ingredients (APIs) such as sildenafil, tadalafil or vardenafil (2 –6) or analogs of these approved APIs (5, 7 –27). Furthermore, products containing multiple PDE-5 inhibitors have been found, both in the literature (5, 10, 18) and in samples analyzed by the US Food and Drug Administration. Currently, several methods are available to determine a single PDE-5 inhibitor or analog (2, 11, 16, 26, 28 – 31), and some are designed to assay more than one analyte (4 – 6, 24, 32 – 34) using either high-performance liquid chromatography with ultraviolet detection or liquid chromatography –mass spectrometry (LC – MS). In cases where certain combinations of PDE-5 inhibitors and/or analogs are present in a single matrix, recently
up to six in one dietary supplement, it may be necessary to run the sample by more than one method to determine the levels of analytes present. A single method able to analyze samples containing multiple PDE-5 inhibitors, and the large number of analogs detected to date, was desired. In this report, a sensitive and accurate method capable of separating 14 PDE-5 inhibitors and analogs in a variety of dosage forms is presented. Figures of merit for the various standards analyzed include linearity, dynamic range, precision, accuracy, limits of detection and quantitation and ruggedness.
Experimental Materials Standard reference material for sildenafil citrate was provided by Pfizer Inc. (New York, NY, USA), tadalafil by Eli Lilly and Company (Indianapolis, IN, USA) and vardenafil hydrochloride trihydrate by Bayer Corporation (West Haven, CT, USA). Standard reference materials for homosildenafil methanesulfonate, hydroxyhomosildenafil citrate and sulfoaildenafil methanesulfonate were available as in-house standards, and having been analytically verified by the Forensic Chemistry Center for use internally. Standards of acetildenafil, aminotadalafil, dimethylsildenafil, hydroxyacetildenafil, hydroxythiohomosildenafil, pseudovardenafil, thiohomosildenafil, thiosildenafil and xanthoanthrafil were purchased from TLC PharmaChem, Inc. (Ontario, Canada). High-performance liquid chromatography (HPLC) grade CH3CN, 0.1% trifluoroacetic acid (TFA) in H2O and 0.1% TFA in CH3CN were purchased from Fisher Scientific (St. Louis, MO, USA). 18 MV cm deionized H2O was generated using a Milli-Q system (Millipore, Billerica, MA, USA). Disposable non-sterile Luer-Lok syringes and 30 mm nylon syringe filters with 0.45 mm pore size were purchased from Fisher Scientific.
Standard preparation Stock standards were prepared at concentrations of 0.2 mg/mL as the free base. Serial dilutions of the stock standard, down to 20 mg/mL, were prepared to create a three-point calibration curve. The standards were made and diluted in CH3CN : H2O (50 : 50, v/v). Multiple analytes may be combined into a single solution or prepared separately. For determination of analogs for which only a limited amount of standard is available, a single solution of the analyte of interest was prepared for the purpose of retention time comparison. To estimate quantity, a calibration curve was prepared using a standard that most closely resembled the analog in structure,
Published by Oxford University Press 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.
ultraviolet (UV) spectral characteristics, and experimentally determined molar absorptivity. These data, as shown in Figures 1 and 2 and Table I, indicate the successful use of a structurally related compound to estimate content.
Sample preparation A representative number of dosage units, generally 5 – 10 capsules or tablets, were combined to create a composite. When
working with a sample that did not have a declared level, a portion equivalent to one-tenth to one-half tablet or capsule content weight was transferred to a suitably sized volumetric flask. Samples having a declared level of API were prepared such that the resulting analyte concentration was 0.1 mg/mL. For both types of samples, the flasks were filled 12 full with CH3CN : H2O (50 : 50, v/v), shaken for 15 min using a mechanical shaker, diluted to volume and mixed. A portion of the extract was filtered.
Figure 1. Structures for the 15 PDE-5 inhibitors and analogs analyzed using the method.
Determination of PDE-5 Inhibitors and Analogs 39
Figure 2. Absorbance spectra for 15 PDE-5 inhibitors and analogs collected using a Hewlett-Packard HP8452A Diode-Array Spectrophotometer. Samples were prepared in solutions of 50 : 50 CH3CN : DI H2O, and concentrations ranged from 4 to 7 mg/mL as the free base.
For fortified samples, an additional portion of sample was prepared in the same manner as the initial sample preparation. Prior to adding solvent, the sample was fortified with an amount of appropriate standard equivalent to 50–150% of the amount determined to be present in the sample and thoroughly mixed to combine. If a sufficient quantity of an analog standard was not available, a standard was chosen that most closely resembled the analog. The fortified sample was then prepared as described above. If necessary, the filtrate was diluted further in order for the peak area response to fall within the calibration curve. 40 Nickum and Flurer
Selection of detection wavelength For the selection of a suitable detection wavelength, standard solutions of 15 PDE-5 inhibitors and analogs were analyzed to determine absorbance. The absorbance spectra were collected using a Hewlett-Packard HP8452A Diode-Array Spectrophotometer (Agilent, Santa Clara, CA, USA). Samples were prepared in solutions of CH3CN : H2O (50 : 50, v/v), and concentrations ranged from 4 to 7 mg/mL (Figure 2). The data show that most have absorbance maxima between 215 and 230 and 280 and 300 nm. Vardenafil and pseudovardenafil do not have maxima in the
region of 280 –300 nm, but they do exhibit absorbance. The region of 285 nm, as opposed to a wavelength in the 215–230 nm range, is selective towards the analytes of interest, and exhibits reduced background absorbance. When necessary, detection at 230 nm allows for increased signal when analyzing low levels of analyte in various samples.
HPLC instrument parameters Analyses were performed using a Waters 2690 Alliance System with a 996 PDA Detector, Empower Software v.2 from Waters Corporation (Milford, MA, USA) and a Zorbax Eclipse XDB-C8, 150 4.6 mm, 5 mm column from Agilent Technologies (Santa Clara, CA, USA). The mobile phase consisted of a 10 min gradient method using Solvent A, 0.1% TFA in H2O, and Solvent B, 0.1% TFA in CH3CN. The gradient was performed as follows: 0 min, 25% B; 0 –7 min, linear to 50% B and 3 min, hold at 50% B. The column was equilibrated at 25% B for 5 min between runs. The flow rate was 1 mL/min, the injection volume was 10 mL and the column temperature was ambient. The detection wavelength was 285 nm, with optional dual detection at 230 nm or online spectral collection from 210 to 400 nm as necessary. If a reference wavelength is used, care in its selection should be taken, as xanthoanthrafil and the thione analogs demonstrate strong absorbance at higher wavelengths (Figure 2).
Method validation The linearity and dynamic range of the method were evaluated using a series of standard solutions. Multiple injections of a wide range of concentrations of vardenafil, sildenafil, tadalafil and the 12 analogs were used to construct calibration curves, and the correlation coefficient, y-intercept and slope were calculated. The average retention time and relative retention time to sildenafil were also determined. Injection precision, limit of detection (LOD) and limit of quantitation (LOQ) for each analyte were determined. One of the system suitability parameters that must be met in a typical United States Pharmacopeia monograph concerns the relative standard
Table I Molar Absorptivities for PDE-5 Inhibitors and Analogsa Name
Vardenafil Pseudovardenafil Hydroxyhomosildenafil Sildenafil Homosildenafil Dimethylsildenafil Hydroxythiohomosildenafil Thiosildenafil Thiohomosildenafil Sulfoaildenafil Aminotadalafil Tadalafil Hydroxyacetildenafil Acetildenafil Xanthoanthrafil a
Determined experimentally, see Figure 2.
deviation (RSD) for replicate injections. To mimic that requirement, a mid-level standard was injected five times. In order to determine LOD and LOQ, a low-level standard was injected seven times, and the standard deviation (SD) was determined. The LOD was defined as three times the SD, and the LOQ as ten times the SD, for each analyte. Method accuracy and precision were assessed using dosage forms of Levitraw, Viagraw and Cialisw. Four preparations of each dosage form were evaluated using the gradient method, and the assay values were compared with those obtained using the manufacturer’s methods for vardenafil, sildenafil and tadalafil. Additional method accuracy data from various dosage forms were determined by comparing assay values to those obtained using the manufacturer’s methods. Ruggedness of the method was determined using Waters, Dionex and Agilent HPLC systems, various C8 columns and multiple analysts. Composites of Levitraw, Viagraw and Cialisw were analyzed, as were three dietary supplement capsule composites that were fortified with homosildenafil, hydroxyhomosildenafil or sulfoaildenafil.
Results Specificity The relative retention times for 15 PDE-5 inhibitors and analogs were determined. Initially, 14 of the analytes were combined into a single mixed standard and analyzed. Table II lists the relative retention time, with respect to sildenafil, for the compounds evaluated. This includes the more recently obtained xanthoanthrafil, which was analyzed individually for inclusion with the mixed standard results. Chromatographic results for the dietary supplement matrix used in the preparation of the homosildenafil, sulfoaildenafil and hydroxyhomosildenafil samples for determination of method ruggedness showed no peaks in the retention time range of the PDE-5 inhibitors and analogs. Additionally, specificity was also demonstrated by the lack of interference from compounds detected in conjunction with PDE-5 inhibitors and analogs, such as caffeine, amino acids, fatty acids, sugar alcohols, synephrine and menthol.
Table II Relative Retention Time for the PDE-5 Inhibitors and Analogs Molar absorptivity (M21 cm21) 285 nm
230 nm
8,611 8,382 13,196 12,604 11,751 11,520 9,783 8,645 9,859 10,291 12,713 13,403 23,708 24,393 3,962
32,752 31,806 31,402 30,161 29,689 27,475 36,211 31,220 33,934 35,043 28,708 29,049 24,537 24,588 17,077
Name
Relative retention time (relative to sildenafil)
Vardenafil Hydroxyacetildenafil Acetildenafil Hydroxyhomosildenafil Sildenafil Homosildenafil Dimethylsildenafil Aminotadalafil Tadalafil Pseudovardenafil Xanthoanthrafila Hydroxythiohomosildenafil Thiosildenafil Thiohomosildenafil Sulfoaildenafil
0.63 0.65 0.70 0.96 1.00 1.05 1.08 1.20 1.41 1.41 1.42 1.49 1.55 1.62 1.64
a A standard for xanthoanthrafil was obtained recently. It was analyzed for retention time information, but it was not included in the mixture evaluated in Figure 3.
Determination of PDE-5 Inhibitors and Analogs 41
Chromatography, linearity, dynamic range, LOD and LOQ Over an extended period of time, multiple concentrations of standards of vardenafil, sildenafil, tadalafil and the 12 analogs were analyzed in duplicate, at minimum, using a single HPLC column. The RSD values for intraday injections are typically ,1.0%. The RSD values for interday analyses are typically ,2.0%, indicating excellent system stability. Calibration curves for the 15 analytes were constructed to determine linearity. Concentrations ranged from 1 to 10 mg/mL for the low standard and 200 to 400 mg/mL for the high standard, and each curve covered at least two orders of magnitude. At least three concentrations of standards were used to create the calibration curve, and each standard was injected a minimum of three times. The correlation coefficient for each curve was calculated to be 0.999 or better. The RSD for injection precision for five injections of a 0.1 mg/mL solution was 0.4% or less for all of the analytes. To determine LOD and LOQ, a 1 mg/mL solution of each of the analytes was injected seven times. The calculated LOD was 0.2 mg/mL or less, and the LOQ was 0.5 mg/mL or less. Accuracy and precision Duplicate preparations of various dosage forms were evaluated by comparing assay values to those obtained using the manufacturer’s methods, as shown in Table III. For sildenafil concentrations
Table III Comparison of Assay Results to Established Methods Analyte
Sildenafil
Tadalafil
Matrix
Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Capsule composite Capsule composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite
Analyte content (mg/dosage) Gradient method
Established methoda,b
52 92 59 97 74 75 60 81 87 53 96 92 96 130 16 15 20 73 17 20 19 19 19 20 7.8 8.8 4.5 9.4 7.1 8.4 7.6 20
50 93 59 99 73 79 61 77 87 52 95 90 96 130 16 15 18 71 17 19 18 19 20 20 8.2 9.1 4.8 10 7.2 8.2 7.5 19
a Established method for sildenafil: “In-Process Revision: Sildenafil Citrate” monograph, Pharmaceutical Forum (USP). b Established method for tadalafil: “Determination of LY450190 (IC351) and Related Substances in Tablet Formulations by Reverse-Phase HPLC”, Eli Lilly and Company Laboratory Procedure B07000, Rev. 3, 14 September 1999.
42 Nickum and Flurer
ranging from 15 to 130 mg per dosage unit, the result from the gradient method was within 94% of the value obtained from the monograph (35). For tadalafil concentrations ranging from 4.5 to 20 mg per dosage unit, the result was within 90% of the manufacturer’s method. Additionally, the gradient method was applied to the three approved dosage forms, and the results were compared with the declared milligram per tablet level. Composites of the approved products were prepared, and four preparations of each composite were analyzed. For Levitraw 20 mg tablets, the average assay value was 20.4 + 0.5 mg (102% declared). For Cialisw 10 mg tablets, the average value was 9.8 + 0.2 mg (98% declared) and for Viagraw 100 mg tablets, the average value was 100 + 1 mg (100% declared). Table IV shows recovery data for samples fortified with various levels of sildenafil, tadalafil and sulfoaildenafil. Recoveries ranged from 87 to 104% for sildenafil, 87 to 107% for tadalafil and 95 to 104% for sulfoaildenafil.
Ruggedness To evaluate method ruggedness, composites of the three approved products and three dietary supplement capsule composites, each containing an analog, were analyzed using separate HPLC systems and multiple analysts, as outlined in Table V. For Viagraw 50 mg tablets, the range in assay values was 47– 51 mg. For Cialisw 20 mg tablets, the range was 19 – 20 mg and for Levitraw 10 mg tablets, the range was 9.3 – 9.9 mg. For a dietary supplement prepared to contain 72 mg homosildenafil per capsule, the range was 71 –75 mg. For a prepared 43 mg sulfoaildenafil per capsule composite, the range was 44– 45 mg, and for a prepared 59 mg hydroxyhomosildenafil per capsule composite, the range was 58 – 65 mg. It was also demonstrated that the assay values at 230 nm were comparable with the values for 285 nm.
Discussion Prior to the development of this method, the determination of sildenafil, tadalafil or vardenafil was performed using individual methods. Sildenafil was determined using the Pharmaceutical Forum monograph for “Sildenafil Tablets” (35). Tadalafil and vardenafil were determined using proprietary methods provided by the manufacturers. Because these methods required the preparation of unique mobile phases and sample diluents, and utilized different chromatographic columns and system parameters, it was problematic to analyze samples containing more than one PDE-5 inhibitor, particularly in cases where the amount of sample was limited. Before the samples were assayed, they were analyzed using LC – MS for identification purposes (3). Using the chromatography parameters of the LC – MS method as a guide, it was determined that a similar method could be used for the quantification of PDE-5 inhibitors and analogs. The resulting gradient program can separate 14 of the 15 PDE-5 inhibitors and analogs investigated within 10 min, as shown in Figure 3. Tadalafil and pseudovardenafil cannot be separated chromatographically using these parameters, but they can be distinguished by their UV spectra. If complete separation is necessary, the
Table IV Spike Recovery Experiments
Table V Evaluation of Method Ruggedness Using Multiple Analysts
Analyte
Matrix
Analyte content (mg/dosage)
Fortification level (mg/dosage)
Sildenafil
Capsule composite Capsule composite Tablet composite Tablet composite Tablet composite Drink mix Bulk powder Tablet composite Tablet composite Sublingual strips Capsule composite Capsule composite Capsule composite Capsule contents Capsule contents Bulk powder Bulk powder Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Suspension Capsule composite Capsule composite Suspension Suspension Capsule composite Capsule contents Capsule composite Capsule composite Capsule composite Capsule composite Capsule composite Capsule composite Capsule composite Capsule composite
150
110
Tadalafil
Sulfoaildenafil
Percent recovery
Analysta
Matrix content as declared
99
160
84
92
87
68
104
100
72
94
99
66
102
33 680 mg/g 16
29 650 mg/g 14
15
21
3.0 mg/strip
3.7 mg/strip
98
60
38
87
110
88
100
78
74
90
110
110
93
110
100
95
370 mg/g 370 mg/g 35
380 mg/g 390 mg/g 23
104 98 103
19
12
102
19
13
87
20
20
102
8.4
13
99
37 mg/mL 23
52 mg/mL 24
99 107
23
21
106
28 mg/mL 29 mg/mL 9.5
29 mg/mL 29 mg/mL 8.9
99 99 104
76
80
98
57
59
95
69
71
101
74
73
104
54
53
101
83
82
101
77
67
101
80
73
95
78
70
99
97 94 98
w
Viagra 50 mg sildenafil per tablet
Analyst 1 Analyst 2 Analyst 3 Averagec SD RSD 95% CI % declared Analyst 1 Analyst 2 Analyst 3 Averagec SD RSD 95% CI % declared Analyst 1 Analyst 2 Analyst 3 Averagec SD RSD 95% CI % declared
Cialisw 20 mg tadalafil per tablet
101
gradient can be extended to 18 min. This extended gradient method also provides greater resolution for the pairs of vardenafil and hydroxyacetildenafil, homosildenafil and dimethylsildenafil and thiohomosildenafil and sulfoaildenafil. In practice, the vast
Levitraw 10 mg vardenafil per tablet
Matrix content as prepared Dietary supplement 72 mg homosildenafil per capsule
Dietary supplement 43 mg sulfoaildenafil per capsule
Dietary supplement 59 mg hydroxyhomosildenafil per capsule
Analyst 1 Analyst 3 Analyst 4 Averagec SD RSD 95% CI % declared Analyst 1 Analyst 2 Analyst 3 Analyst 4 Averagec SD RSD 95% CI % declared Analyst 1 Analyst 2 Analyst 3 Analyst 4 Averagec SD RSD 95% CI % declared
Quantity found (mgb) 230 nm
285 nm
46.8 51.2 49.8 49.3 2.2 4.4 2.3 98.6 20.2 19.4 19.6 19.7 0.4 2.0 0.4 98.5 9.35 9.94 9.51 9.60 0.3 2.8 0.3 96.0
46.7 51.2 49.9 49.3 2.2 4.4 2.3 98.6 20.1 19.3 19.5 19.6 0.4 2.0 0.4 98.0 9.34 9.94 9.59 9.62 0.3 2.9 0.3 96.2
75.3 74.3 71.3 73.6 3.3 4.5 3.5 102 44.4 44.6 44.7 44.1 44.4 0.5 1.2 0.4 103 65.2 64.0 59.2 58.3 61.7 3.2 5.3 2.7 105
74.8 74.5 71.3 73.5 3.3 4.4 3.4 102 44.4 44.4 44.6 44.4 44.4 0.5 1.1 0.4 103 65.1 63.8 59.3 58.1 61.6 3.3 5.4 2.8 104
a Analyst 1: Waters 2690 Alliance, 996 PDA detector; Zorbax Eclipse XDB-C8, 4.6 150 mm. Analyst 2: Waters 2690 Alliance, 996 PDA detector; Zorbax Eclipse XDB-C8, 4.6 150 mm. Analyst 3: Dionex Summit, PDA 100 detector; Waters XTerra RP8, 4.6 150 mm. Analyst 4: Agilent 1200 Series HPLC, DAD detector; Zorbax SB-C8, 4.6 150 mm. b Duplicate preparations of each matrix were analyzed. The value reported is the average of the two preparations. c The values for average, SD, RSD, 95% confidence interval (CI) and percent declared were calculated using all analysts’ preparations.
majority of samples analyzed do not contain the closely retained pairs, so the shorter gradient method is sufficient and generally recommended. The PDE-5 inhibitors and analogs analyzed were grouped into six distinct classes based on their UV spectra, as shown in Figure 2. Using these classes, suitable standards for estimation, when the specific analyte is not available or is cost prohibitive for infrequent analysis, can be selected more easily. Additionally, if a Determination of PDE-5 Inhibitors and Analogs 43
Figure 3. (a) Chromatogram of 14 PDE-5 inhibitors and analogs, using the method as described. (1) Vardenafil, (2) hydroxyacetildenafil, (3) acetildenafil, (4) hydroxyhomosildenafil, (5) sildenafil, (6) homosildenafil, (7) dimethylsildenafil, (8) aminotadalafil, (9) tadalafil, (10) pseudovardenafil, (11) hydroxythiohomosildenafil, (12) thiosildenafil, (13) thiohomosildenafil and (14) sulfoaildenafil. Note that tadalafil and pseudovardenafil coelute. Concentration values for the free base ranged from 28 mg/mL for acetildenafil to 135 mg/mL for thiosildenafil. (b) Chromatogram of 14 PDE-5 inhibitors and analogs, using the extended gradient method. The extended gradient program consisted of an 18 min run using Solvent A, 0.1% TFA in H2O, and Solvent B, 0.1% TFA in CH3CN. The gradient was performed as follows: 0 –2 min, 25% B; 2 –17 min, linear to 50% B and 1 min, hold at 50% B. All other experimental parameters were the same.
standard for a particular analog was not available, peak identification may be determined by UV spectrum and LC – MS elution order comparisons. Conclusion This method provides separation of 14 PDE-5 inhibitors and analogs in a variety of forms, including tablets, capsules, bulk powders, troches and liquids, in 10 min. It is linear over a wide range of concentrations, sensitive to trace levels, accurate in dosage forms as well as supplements and rugged across analysts and instruments. Additionally, by expanding the gradient method to an 18-min run-time, all 15 compounds can be resolved, offering 44 Nickum and Flurer
the flexibility needed to separate and analyze new analogs as they are encountered. Additionally, UV data presented indicate that PDE-5 inhibitors with similar structures have similar spectra and similar molar absorptivities. This proved useful in the latest characterization of two new tadalafil analogs, 2-hydroxypropylnortadalafil and n-butylnortadalafil (36), as they have spectra similar to tadalafil. As the work in this field of study continues, it may be difficult to maintain an up-to-date collection of each standard needed to perform quantitative analysis and the ability to use a spectrally related compound to provide estimated levels is helpful in cases where the procurement of a standard is difficult, time consuming or cost prohibitive.
Acknowledgments The author would like to thank Merrie P. Jackson, Valerie M. Toomey, Samuel R. Gratz, PhD and Sara E. Robison, PhD for their assistance and contributions.
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Determination of PDE-5 Inhibitors and Analogs 45
32. De Orsi, D., Pellegrini, M., Marchei, E., Nebuloni, P., Gallinella, B., Scaravelli, G., et al.; High performance liquid chromatography-diode array and electrospray-mass spectrometry analysis of vardenafil, sildenafil, tadalafil, testosterone and local anesthetics in cosmetic creams sold on the Internet web sites; Journal of Pharmaceutical and Biomedical Analysis, (2009); 50: 362– 369. 33. Venhuis, B.J., Zomer, G., Vredenbregt, M.J., de Kaste, D.; The identification of ( – )-trans-tadalafil, tadalafil, and sildenafil in counterfeit Cialis w and the optical purity of tadalafil stereoisomers; Journal of Pharmaceutical and Biomedical Analysis, (2010); 51: 723 – 727.
46 Nickum and Flurer
34. Sacre´, P.Y., Deconinck, E., Chiap, P., Crommen, J., Mansion, F., Rozet, E., et al.; Development and validation of a ultra-high-performance liquid chromatography-UV method for the detection and quantification of erectile dysfunction drugs and some of their analogues found in counterfeit medicines; Journal of Chromatography A, (2011); 1218: 6439–6447. 35. Sildenafil Citrate/Sildenafil Tablets; Pharmacopeial Forum, (1998); 24: 7182–7185. 36. Toomey, V.M., Litzau, J.J., Flurer, C.L.; Isolation and structural characterization of two tadalafil analogs found in dietary supplements; Journal of Pharmaceutical and Biomedical Analysis, (2012); 59: 50–57.
Article
Determination of Phosphodiesterase-5 Inhibitors and Analogs Using High-Performance Liquid Chromatography with Ultraviolet Detection Elisa A. Nickum* and Cheryl L. Flurer U.S. Food and Drug Administration, Forensic Chemistry Center, 6751 Steger Drive, Cincinnati, OH 45237, USA * Author to whom correspondence should be addressed. Email: [email protected] Received 16 July 2013; revised 10 January 2014
A considerable number of erectile dysfunction products, and dietary supplements suspected of containing phosphodiesterase-5 (PDE-5) inhibitors, have been analyzed by the US Food and Drug Administration. Often these samples are found to contain the approved active pharmaceutical ingredients (APIs) such as sildenafil, tadalafil or vardenafil. However, analogs of these APIs have also been identified in many samples and products containing multiple PDE-5 inhibitors have also been found. A single high-performance liquid chromatography with ultraviolet detection method has been developed for the determination of sildenafil, tadalafil, vardenafil and a number of commonly encountered analogs in pharmaceutical dosage forms and dietary supplement products, including tablets, capsules, bulk powders, troches and liquids. This method was designed as an alternative to methods developed for the determination of a single PDE-5 inhibitor. Using this protocol, 14 PDE-5 inhibitor compounds can be separated and determined in a single analysis.
Introduction The class of drugs known as phosphodiesterase-5 (PDE-5) inhibitors includes sildenafil citrate (Viagraw, Pfizer), tadalafil (Cialisw, Eli Lilly) and vardenafil hydrochloride trihydrate (Levitraw, Bayer), and is marketed and approved for use in the USA in the treatment of erectile dysfunction (ED). During the last decade, these oral drugs have been the first-line treatment for ED as they are easily administered, relatively non-invasive, reversible and well tolerated. However, taking these drugs can be dangerous to those who suffer from certain comorbid conditions such as hypertension, heart disease or diabetes, or those who take nitrates, a-blockers or blood-thinning medication (1). As a result, many men seek alternative medicines, in the form of herbal and dietary supplements, as a therapy for ED with the belief that these products are safe. Studies indicate that these products are often found to contain the active pharmaceutical ingredients (APIs) such as sildenafil, tadalafil or vardenafil (2 –6) or analogs of these approved APIs (5, 7 –27). Furthermore, products containing multiple PDE-5 inhibitors have been found, both in the literature (5, 10, 18) and in samples analyzed by the US Food and Drug Administration. Currently, several methods are available to determine a single PDE-5 inhibitor or analog (2, 11, 16, 26, 28 – 31), and some are designed to assay more than one analyte (4 – 6, 24, 32 – 34) using either high-performance liquid chromatography with ultraviolet detection or liquid chromatography –mass spectrometry (LC – MS). In cases where certain combinations of PDE-5 inhibitors and/or analogs are present in a single matrix, recently
up to six in one dietary supplement, it may be necessary to run the sample by more than one method to determine the levels of analytes present. A single method able to analyze samples containing multiple PDE-5 inhibitors, and the large number of analogs detected to date, was desired. In this report, a sensitive and accurate method capable of separating 14 PDE-5 inhibitors and analogs in a variety of dosage forms is presented. Figures of merit for the various standards analyzed include linearity, dynamic range, precision, accuracy, limits of detection and quantitation and ruggedness.
Experimental Materials Standard reference material for sildenafil citrate was provided by Pfizer Inc. (New York, NY, USA), tadalafil by Eli Lilly and Company (Indianapolis, IN, USA) and vardenafil hydrochloride trihydrate by Bayer Corporation (West Haven, CT, USA). Standard reference materials for homosildenafil methanesulfonate, hydroxyhomosildenafil citrate and sulfoaildenafil methanesulfonate were available as in-house standards, and having been analytically verified by the Forensic Chemistry Center for use internally. Standards of acetildenafil, aminotadalafil, dimethylsildenafil, hydroxyacetildenafil, hydroxythiohomosildenafil, pseudovardenafil, thiohomosildenafil, thiosildenafil and xanthoanthrafil were purchased from TLC PharmaChem, Inc. (Ontario, Canada). High-performance liquid chromatography (HPLC) grade CH3CN, 0.1% trifluoroacetic acid (TFA) in H2O and 0.1% TFA in CH3CN were purchased from Fisher Scientific (St. Louis, MO, USA). 18 MV cm deionized H2O was generated using a Milli-Q system (Millipore, Billerica, MA, USA). Disposable non-sterile Luer-Lok syringes and 30 mm nylon syringe filters with 0.45 mm pore size were purchased from Fisher Scientific.
Standard preparation Stock standards were prepared at concentrations of 0.2 mg/mL as the free base. Serial dilutions of the stock standard, down to 20 mg/mL, were prepared to create a three-point calibration curve. The standards were made and diluted in CH3CN : H2O (50 : 50, v/v). Multiple analytes may be combined into a single solution or prepared separately. For determination of analogs for which only a limited amount of standard is available, a single solution of the analyte of interest was prepared for the purpose of retention time comparison. To estimate quantity, a calibration curve was prepared using a standard that most closely resembled the analog in structure,
Published by Oxford University Press 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.
ultraviolet (UV) spectral characteristics, and experimentally determined molar absorptivity. These data, as shown in Figures 1 and 2 and Table I, indicate the successful use of a structurally related compound to estimate content.
Sample preparation A representative number of dosage units, generally 5 – 10 capsules or tablets, were combined to create a composite. When
working with a sample that did not have a declared level, a portion equivalent to one-tenth to one-half tablet or capsule content weight was transferred to a suitably sized volumetric flask. Samples having a declared level of API were prepared such that the resulting analyte concentration was 0.1 mg/mL. For both types of samples, the flasks were filled 12 full with CH3CN : H2O (50 : 50, v/v), shaken for 15 min using a mechanical shaker, diluted to volume and mixed. A portion of the extract was filtered.
Figure 1. Structures for the 15 PDE-5 inhibitors and analogs analyzed using the method.
Determination of PDE-5 Inhibitors and Analogs 39
Figure 2. Absorbance spectra for 15 PDE-5 inhibitors and analogs collected using a Hewlett-Packard HP8452A Diode-Array Spectrophotometer. Samples were prepared in solutions of 50 : 50 CH3CN : DI H2O, and concentrations ranged from 4 to 7 mg/mL as the free base.
For fortified samples, an additional portion of sample was prepared in the same manner as the initial sample preparation. Prior to adding solvent, the sample was fortified with an amount of appropriate standard equivalent to 50–150% of the amount determined to be present in the sample and thoroughly mixed to combine. If a sufficient quantity of an analog standard was not available, a standard was chosen that most closely resembled the analog. The fortified sample was then prepared as described above. If necessary, the filtrate was diluted further in order for the peak area response to fall within the calibration curve. 40 Nickum and Flurer
Selection of detection wavelength For the selection of a suitable detection wavelength, standard solutions of 15 PDE-5 inhibitors and analogs were analyzed to determine absorbance. The absorbance spectra were collected using a Hewlett-Packard HP8452A Diode-Array Spectrophotometer (Agilent, Santa Clara, CA, USA). Samples were prepared in solutions of CH3CN : H2O (50 : 50, v/v), and concentrations ranged from 4 to 7 mg/mL (Figure 2). The data show that most have absorbance maxima between 215 and 230 and 280 and 300 nm. Vardenafil and pseudovardenafil do not have maxima in the
region of 280 –300 nm, but they do exhibit absorbance. The region of 285 nm, as opposed to a wavelength in the 215–230 nm range, is selective towards the analytes of interest, and exhibits reduced background absorbance. When necessary, detection at 230 nm allows for increased signal when analyzing low levels of analyte in various samples.
HPLC instrument parameters Analyses were performed using a Waters 2690 Alliance System with a 996 PDA Detector, Empower Software v.2 from Waters Corporation (Milford, MA, USA) and a Zorbax Eclipse XDB-C8, 150 4.6 mm, 5 mm column from Agilent Technologies (Santa Clara, CA, USA). The mobile phase consisted of a 10 min gradient method using Solvent A, 0.1% TFA in H2O, and Solvent B, 0.1% TFA in CH3CN. The gradient was performed as follows: 0 min, 25% B; 0 –7 min, linear to 50% B and 3 min, hold at 50% B. The column was equilibrated at 25% B for 5 min between runs. The flow rate was 1 mL/min, the injection volume was 10 mL and the column temperature was ambient. The detection wavelength was 285 nm, with optional dual detection at 230 nm or online spectral collection from 210 to 400 nm as necessary. If a reference wavelength is used, care in its selection should be taken, as xanthoanthrafil and the thione analogs demonstrate strong absorbance at higher wavelengths (Figure 2).
Method validation The linearity and dynamic range of the method were evaluated using a series of standard solutions. Multiple injections of a wide range of concentrations of vardenafil, sildenafil, tadalafil and the 12 analogs were used to construct calibration curves, and the correlation coefficient, y-intercept and slope were calculated. The average retention time and relative retention time to sildenafil were also determined. Injection precision, limit of detection (LOD) and limit of quantitation (LOQ) for each analyte were determined. One of the system suitability parameters that must be met in a typical United States Pharmacopeia monograph concerns the relative standard
Table I Molar Absorptivities for PDE-5 Inhibitors and Analogsa Name
Vardenafil Pseudovardenafil Hydroxyhomosildenafil Sildenafil Homosildenafil Dimethylsildenafil Hydroxythiohomosildenafil Thiosildenafil Thiohomosildenafil Sulfoaildenafil Aminotadalafil Tadalafil Hydroxyacetildenafil Acetildenafil Xanthoanthrafil a
Determined experimentally, see Figure 2.
deviation (RSD) for replicate injections. To mimic that requirement, a mid-level standard was injected five times. In order to determine LOD and LOQ, a low-level standard was injected seven times, and the standard deviation (SD) was determined. The LOD was defined as three times the SD, and the LOQ as ten times the SD, for each analyte. Method accuracy and precision were assessed using dosage forms of Levitraw, Viagraw and Cialisw. Four preparations of each dosage form were evaluated using the gradient method, and the assay values were compared with those obtained using the manufacturer’s methods for vardenafil, sildenafil and tadalafil. Additional method accuracy data from various dosage forms were determined by comparing assay values to those obtained using the manufacturer’s methods. Ruggedness of the method was determined using Waters, Dionex and Agilent HPLC systems, various C8 columns and multiple analysts. Composites of Levitraw, Viagraw and Cialisw were analyzed, as were three dietary supplement capsule composites that were fortified with homosildenafil, hydroxyhomosildenafil or sulfoaildenafil.
Results Specificity The relative retention times for 15 PDE-5 inhibitors and analogs were determined. Initially, 14 of the analytes were combined into a single mixed standard and analyzed. Table II lists the relative retention time, with respect to sildenafil, for the compounds evaluated. This includes the more recently obtained xanthoanthrafil, which was analyzed individually for inclusion with the mixed standard results. Chromatographic results for the dietary supplement matrix used in the preparation of the homosildenafil, sulfoaildenafil and hydroxyhomosildenafil samples for determination of method ruggedness showed no peaks in the retention time range of the PDE-5 inhibitors and analogs. Additionally, specificity was also demonstrated by the lack of interference from compounds detected in conjunction with PDE-5 inhibitors and analogs, such as caffeine, amino acids, fatty acids, sugar alcohols, synephrine and menthol.
Table II Relative Retention Time for the PDE-5 Inhibitors and Analogs Molar absorptivity (M21 cm21) 285 nm
230 nm
8,611 8,382 13,196 12,604 11,751 11,520 9,783 8,645 9,859 10,291 12,713 13,403 23,708 24,393 3,962
32,752 31,806 31,402 30,161 29,689 27,475 36,211 31,220 33,934 35,043 28,708 29,049 24,537 24,588 17,077
Name
Relative retention time (relative to sildenafil)
Vardenafil Hydroxyacetildenafil Acetildenafil Hydroxyhomosildenafil Sildenafil Homosildenafil Dimethylsildenafil Aminotadalafil Tadalafil Pseudovardenafil Xanthoanthrafila Hydroxythiohomosildenafil Thiosildenafil Thiohomosildenafil Sulfoaildenafil
0.63 0.65 0.70 0.96 1.00 1.05 1.08 1.20 1.41 1.41 1.42 1.49 1.55 1.62 1.64
a A standard for xanthoanthrafil was obtained recently. It was analyzed for retention time information, but it was not included in the mixture evaluated in Figure 3.
Determination of PDE-5 Inhibitors and Analogs 41
Chromatography, linearity, dynamic range, LOD and LOQ Over an extended period of time, multiple concentrations of standards of vardenafil, sildenafil, tadalafil and the 12 analogs were analyzed in duplicate, at minimum, using a single HPLC column. The RSD values for intraday injections are typically ,1.0%. The RSD values for interday analyses are typically ,2.0%, indicating excellent system stability. Calibration curves for the 15 analytes were constructed to determine linearity. Concentrations ranged from 1 to 10 mg/mL for the low standard and 200 to 400 mg/mL for the high standard, and each curve covered at least two orders of magnitude. At least three concentrations of standards were used to create the calibration curve, and each standard was injected a minimum of three times. The correlation coefficient for each curve was calculated to be 0.999 or better. The RSD for injection precision for five injections of a 0.1 mg/mL solution was 0.4% or less for all of the analytes. To determine LOD and LOQ, a 1 mg/mL solution of each of the analytes was injected seven times. The calculated LOD was 0.2 mg/mL or less, and the LOQ was 0.5 mg/mL or less. Accuracy and precision Duplicate preparations of various dosage forms were evaluated by comparing assay values to those obtained using the manufacturer’s methods, as shown in Table III. For sildenafil concentrations
Table III Comparison of Assay Results to Established Methods Analyte
Sildenafil
Tadalafil
Matrix
Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Capsule composite Capsule composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite
Analyte content (mg/dosage) Gradient method
Established methoda,b
52 92 59 97 74 75 60 81 87 53 96 92 96 130 16 15 20 73 17 20 19 19 19 20 7.8 8.8 4.5 9.4 7.1 8.4 7.6 20
50 93 59 99 73 79 61 77 87 52 95 90 96 130 16 15 18 71 17 19 18 19 20 20 8.2 9.1 4.8 10 7.2 8.2 7.5 19
a Established method for sildenafil: “In-Process Revision: Sildenafil Citrate” monograph, Pharmaceutical Forum (USP). b Established method for tadalafil: “Determination of LY450190 (IC351) and Related Substances in Tablet Formulations by Reverse-Phase HPLC”, Eli Lilly and Company Laboratory Procedure B07000, Rev. 3, 14 September 1999.
42 Nickum and Flurer
ranging from 15 to 130 mg per dosage unit, the result from the gradient method was within 94% of the value obtained from the monograph (35). For tadalafil concentrations ranging from 4.5 to 20 mg per dosage unit, the result was within 90% of the manufacturer’s method. Additionally, the gradient method was applied to the three approved dosage forms, and the results were compared with the declared milligram per tablet level. Composites of the approved products were prepared, and four preparations of each composite were analyzed. For Levitraw 20 mg tablets, the average assay value was 20.4 + 0.5 mg (102% declared). For Cialisw 10 mg tablets, the average value was 9.8 + 0.2 mg (98% declared) and for Viagraw 100 mg tablets, the average value was 100 + 1 mg (100% declared). Table IV shows recovery data for samples fortified with various levels of sildenafil, tadalafil and sulfoaildenafil. Recoveries ranged from 87 to 104% for sildenafil, 87 to 107% for tadalafil and 95 to 104% for sulfoaildenafil.
Ruggedness To evaluate method ruggedness, composites of the three approved products and three dietary supplement capsule composites, each containing an analog, were analyzed using separate HPLC systems and multiple analysts, as outlined in Table V. For Viagraw 50 mg tablets, the range in assay values was 47– 51 mg. For Cialisw 20 mg tablets, the range was 19 – 20 mg and for Levitraw 10 mg tablets, the range was 9.3 – 9.9 mg. For a dietary supplement prepared to contain 72 mg homosildenafil per capsule, the range was 71 –75 mg. For a prepared 43 mg sulfoaildenafil per capsule composite, the range was 44– 45 mg, and for a prepared 59 mg hydroxyhomosildenafil per capsule composite, the range was 58 – 65 mg. It was also demonstrated that the assay values at 230 nm were comparable with the values for 285 nm.
Discussion Prior to the development of this method, the determination of sildenafil, tadalafil or vardenafil was performed using individual methods. Sildenafil was determined using the Pharmaceutical Forum monograph for “Sildenafil Tablets” (35). Tadalafil and vardenafil were determined using proprietary methods provided by the manufacturers. Because these methods required the preparation of unique mobile phases and sample diluents, and utilized different chromatographic columns and system parameters, it was problematic to analyze samples containing more than one PDE-5 inhibitor, particularly in cases where the amount of sample was limited. Before the samples were assayed, they were analyzed using LC – MS for identification purposes (3). Using the chromatography parameters of the LC – MS method as a guide, it was determined that a similar method could be used for the quantification of PDE-5 inhibitors and analogs. The resulting gradient program can separate 14 of the 15 PDE-5 inhibitors and analogs investigated within 10 min, as shown in Figure 3. Tadalafil and pseudovardenafil cannot be separated chromatographically using these parameters, but they can be distinguished by their UV spectra. If complete separation is necessary, the
Table IV Spike Recovery Experiments
Table V Evaluation of Method Ruggedness Using Multiple Analysts
Analyte
Matrix
Analyte content (mg/dosage)
Fortification level (mg/dosage)
Sildenafil
Capsule composite Capsule composite Tablet composite Tablet composite Tablet composite Drink mix Bulk powder Tablet composite Tablet composite Sublingual strips Capsule composite Capsule composite Capsule composite Capsule contents Capsule contents Bulk powder Bulk powder Tablet composite Tablet composite Tablet composite Tablet composite Tablet composite Suspension Capsule composite Capsule composite Suspension Suspension Capsule composite Capsule contents Capsule composite Capsule composite Capsule composite Capsule composite Capsule composite Capsule composite Capsule composite Capsule composite
150
110
Tadalafil
Sulfoaildenafil
Percent recovery
Analysta
Matrix content as declared
99
160
84
92
87
68
104
100
72
94
99
66
102
33 680 mg/g 16
29 650 mg/g 14
15
21
3.0 mg/strip
3.7 mg/strip
98
60
38
87
110
88
100
78
74
90
110
110
93
110
100
95
370 mg/g 370 mg/g 35
380 mg/g 390 mg/g 23
104 98 103
19
12
102
19
13
87
20
20
102
8.4
13
99
37 mg/mL 23
52 mg/mL 24
99 107
23
21
106
28 mg/mL 29 mg/mL 9.5
29 mg/mL 29 mg/mL 8.9
99 99 104
76
80
98
57
59
95
69
71
101
74
73
104
54
53
101
83
82
101
77
67
101
80
73
95
78
70
99
97 94 98
w
Viagra 50 mg sildenafil per tablet
Analyst 1 Analyst 2 Analyst 3 Averagec SD RSD 95% CI % declared Analyst 1 Analyst 2 Analyst 3 Averagec SD RSD 95% CI % declared Analyst 1 Analyst 2 Analyst 3 Averagec SD RSD 95% CI % declared
Cialisw 20 mg tadalafil per tablet
101
gradient can be extended to 18 min. This extended gradient method also provides greater resolution for the pairs of vardenafil and hydroxyacetildenafil, homosildenafil and dimethylsildenafil and thiohomosildenafil and sulfoaildenafil. In practice, the vast
Levitraw 10 mg vardenafil per tablet
Matrix content as prepared Dietary supplement 72 mg homosildenafil per capsule
Dietary supplement 43 mg sulfoaildenafil per capsule
Dietary supplement 59 mg hydroxyhomosildenafil per capsule
Analyst 1 Analyst 3 Analyst 4 Averagec SD RSD 95% CI % declared Analyst 1 Analyst 2 Analyst 3 Analyst 4 Averagec SD RSD 95% CI % declared Analyst 1 Analyst 2 Analyst 3 Analyst 4 Averagec SD RSD 95% CI % declared
Quantity found (mgb) 230 nm
285 nm
46.8 51.2 49.8 49.3 2.2 4.4 2.3 98.6 20.2 19.4 19.6 19.7 0.4 2.0 0.4 98.5 9.35 9.94 9.51 9.60 0.3 2.8 0.3 96.0
46.7 51.2 49.9 49.3 2.2 4.4 2.3 98.6 20.1 19.3 19.5 19.6 0.4 2.0 0.4 98.0 9.34 9.94 9.59 9.62 0.3 2.9 0.3 96.2
75.3 74.3 71.3 73.6 3.3 4.5 3.5 102 44.4 44.6 44.7 44.1 44.4 0.5 1.2 0.4 103 65.2 64.0 59.2 58.3 61.7 3.2 5.3 2.7 105
74.8 74.5 71.3 73.5 3.3 4.4 3.4 102 44.4 44.4 44.6 44.4 44.4 0.5 1.1 0.4 103 65.1 63.8 59.3 58.1 61.6 3.3 5.4 2.8 104
a Analyst 1: Waters 2690 Alliance, 996 PDA detector; Zorbax Eclipse XDB-C8, 4.6 150 mm. Analyst 2: Waters 2690 Alliance, 996 PDA detector; Zorbax Eclipse XDB-C8, 4.6 150 mm. Analyst 3: Dionex Summit, PDA 100 detector; Waters XTerra RP8, 4.6 150 mm. Analyst 4: Agilent 1200 Series HPLC, DAD detector; Zorbax SB-C8, 4.6 150 mm. b Duplicate preparations of each matrix were analyzed. The value reported is the average of the two preparations. c The values for average, SD, RSD, 95% confidence interval (CI) and percent declared were calculated using all analysts’ preparations.
majority of samples analyzed do not contain the closely retained pairs, so the shorter gradient method is sufficient and generally recommended. The PDE-5 inhibitors and analogs analyzed were grouped into six distinct classes based on their UV spectra, as shown in Figure 2. Using these classes, suitable standards for estimation, when the specific analyte is not available or is cost prohibitive for infrequent analysis, can be selected more easily. Additionally, if a Determination of PDE-5 Inhibitors and Analogs 43
Figure 3. (a) Chromatogram of 14 PDE-5 inhibitors and analogs, using the method as described. (1) Vardenafil, (2) hydroxyacetildenafil, (3) acetildenafil, (4) hydroxyhomosildenafil, (5) sildenafil, (6) homosildenafil, (7) dimethylsildenafil, (8) aminotadalafil, (9) tadalafil, (10) pseudovardenafil, (11) hydroxythiohomosildenafil, (12) thiosildenafil, (13) thiohomosildenafil and (14) sulfoaildenafil. Note that tadalafil and pseudovardenafil coelute. Concentration values for the free base ranged from 28 mg/mL for acetildenafil to 135 mg/mL for thiosildenafil. (b) Chromatogram of 14 PDE-5 inhibitors and analogs, using the extended gradient method. The extended gradient program consisted of an 18 min run using Solvent A, 0.1% TFA in H2O, and Solvent B, 0.1% TFA in CH3CN. The gradient was performed as follows: 0 –2 min, 25% B; 2 –17 min, linear to 50% B and 1 min, hold at 50% B. All other experimental parameters were the same.
standard for a particular analog was not available, peak identification may be determined by UV spectrum and LC – MS elution order comparisons. Conclusion This method provides separation of 14 PDE-5 inhibitors and analogs in a variety of forms, including tablets, capsules, bulk powders, troches and liquids, in 10 min. It is linear over a wide range of concentrations, sensitive to trace levels, accurate in dosage forms as well as supplements and rugged across analysts and instruments. Additionally, by expanding the gradient method to an 18-min run-time, all 15 compounds can be resolved, offering 44 Nickum and Flurer
the flexibility needed to separate and analyze new analogs as they are encountered. Additionally, UV data presented indicate that PDE-5 inhibitors with similar structures have similar spectra and similar molar absorptivities. This proved useful in the latest characterization of two new tadalafil analogs, 2-hydroxypropylnortadalafil and n-butylnortadalafil (36), as they have spectra similar to tadalafil. As the work in this field of study continues, it may be difficult to maintain an up-to-date collection of each standard needed to perform quantitative analysis and the ability to use a spectrally related compound to provide estimated levels is helpful in cases where the procurement of a standard is difficult, time consuming or cost prohibitive.
Acknowledgments The author would like to thank Merrie P. Jackson, Valerie M. Toomey, Samuel R. Gratz, PhD and Sara E. Robison, PhD for their assistance and contributions.
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