usp–nf | usp-nf - revision bulletin....bulk ingredients • (rb 1-aug-2016) and dosage forms tated...

<2251> Adulteration of Dietary Supplements with Drugs and Drug Analogs (superseded title) <2251> Screening for Undeclared Drugs and Drug Analogues (new title) Type of Posting Revision Bulletin Posting Date 27–May–2016 Official Date 01–Aug–2016 Expert Committee Non-Botanical Dietary Supplements Reason for Revision Compliance In accordance with the Rules and Procedures of the 2015-2020 Council of Experts, the Non-Botanical Dietary Supplement Expert Committee has revised the General Chapter <2251> Adulteration of Dietary Supplements with Drugs and Drug Analogs. The purpose of the revision is to:

• Align the General Chapter terminology with that currently in use by the FDA • Update of the General Chapter title to better reflect its content. The revised title

is “Screening for Undeclared Drugs and Drug Analogues” • Update the Chemical Abstracts (CAS) numbers for compounds in Tables 4 and 5

The Adulteration of Dietary Supplements with Drugs and Drug Analogs Revision Bulletin supersedes the Adulteration of Dietary Supplements with Drugs and Drug Analogs General Chapter published in the First Supplement to USP 39–NF 34, and will become official on August 1, 2016. The Revision Bulletin will be incorporated in the USP 40–NF 35. Should you have any questions, please contact Anton Bzhelyansky, Scientific Liaison, at (301) 203–6303 or [email protected].

C173956_160319-M8206-NBDS2015, Rev. 0 20160527

mailto:[email protected]

Revision BulletinOfficial August 1, 2016 ⟨2251⟩ Screening for Undeclared Drugs and Drug Analogues 1

functionally coherent group of adulterants, including sev-Add the following: eral approved drugs, their numerous approved and unap-

proved analogues, and synthetic intermediates. •.• (RB 1-Aug-

2016) Their functionality is manifested by inhibition ofphosphodiesterase type 5 enzyme (PDE5), which hydro-Change to read: lyzes cyclic guanosine 3′,5′-monophosphate (cGMP); thisgroup of •.compounds• (RB 1-Aug-2016) is frequently identifiedas PDE5 inhibitors. Screening methods for •.products• (RB

.

1-Aug-2016) adulterated with ED •.compounds• (RB 1-Aug-2016) are■

.⟨2251⟩ •.SCREENING FOR UNDECLARED presented in Appendix A.• Weight Loss (WL): This category comprises a function-DRUGS AND DRUG ANALOGUES• (RB 1-Aug-

ally and chemically diverse collection of compounds that2016) include stimulants, laxatives, diuretics, anorexiants, andpsychoactive drugs. Although stimulants constitute anINTRODUCTION important segment of WL adulterants, the oral anorexiantsibutramine dominates this category, frequently in com-

Change to read: bination with phenolphthalein, a laxative. Methods foranalysis of •.products• (RB 1-Aug-2016) adulterated with WL •.

compounds• (RB 1-Aug-2016) will be addressed in Appendix BThe illegal addition of undeclared synthetic compounds to(to come).products marketed as dietary supplements1

. (DS) is •.an issue• Sports Performance Enhancement (SPE): These com-of universal concern.• (RB 1-Aug-2016) This fraud is practiced to

pounds constitute the third major category of adultera-impart therapeutic effects that cannot be achieved by the •.

tion. Professional and amateur athletes are targeted withdietary ingredients• (RB 1-Aug-2016) alone. Increasingly, syntheticdesigner anabolic steroids and stimulants, which are sys-intermediates and structural analogues of the pharmaceuti-tematically banned by the World Anti-Doping Agency.cals and drugs that have been discontinued or withdrawnFunctional and structural diversity, synthetic proclivity offrom the market •.• (RB 1-Aug-2016) are being used as adulterants.the adulterators, and the generally small amounts of theMultiple adulterating compounds may be added to a singleinfringing substances required to elicit a therapeutic ef-•

.product,• (RB 1-Aug-2016) frequently in erratic amounts. fect make this category especially challenging to address.The proposed test methodologies facilitate screening •.• (RB 1-Aug-

These supplements are customarily formulated in protein-2016) for synthetic adulterants. No individual technique is ca-and fat-rich matrices, thereby further complicating detec-pable of addressing all potential analytes; thus, a combina-tion. For these reasons, GC- and LC-MSn

. techniques con-tion of orthogonal approaches adds certainty to the analyti-stitute primary analytical methodologies within this cate-cal outcome. Mass spectrometric techniques provide stronggory. Analysis of •.products• (RB 1-Aug-2016) adulterated withsubstantiation of the analytical findings. In some cases, e.g.,SPE •.compounds• (RB 1-Aug-2016) will be addressed in Appen-with hormonal drugs, the amounts of physiologically rele-dix C (to come).vant adulterants may be so low that GC-MS or LC-MS may

be the only fitting analytical options.The express purpose of assembling the procedures recom- Change to read:mended herein is their •.suitability for• (RB 1-Aug-2016) screening.

The level of evidence achievable by application of one or •.BULK INGREDIENTS• (RB 1-Aug-2016) AND DOSAGE FORMSseveral of the recommended procedures is ultimately dic- •

.Adulteration may occur either at the level of bulk ingredi-tated by the specific requirements of the end-user. It should ents• (RB 1-Aug-2016) or at any •.subsequent• (RB 1-Aug-2016) stagebe noted that structure elucidation and quantitative assess- of the finished product manufacturing. Analysts should bement •.• (RB 1-Aug-2016) are beyond the scope of this chapter. mindful of the •.possibility of• (RB 1-Aug-2016) adulterants •.This chapter is meant to be updated regularly, •.as• (RB 1-Aug-2016) physically associated with• (RB 1-Aug-2016) the finished dosagenew concealment methodologies for the adulterants are in- •.• (RB 1-Aug-2016) matrix or excipients, as well as components.troduced, or improvements to the methods of analysis are In the latter, synthetic compounds •.have been found• (RB 1-realized.

Aug-2016) embedded into the capsule shell body. This under-scores the need for deliberate adjustment to the laboratory

Change to read: procedures that typically focus on the capsule contentsalone. Appropriate sampling practices for powders and fin-ished dosage forms should be exercised, particularly when•

.• (RB 1-Aug-2016) ADULTERATION CATEGORIESonly a limited amount of sample is available.The following major categories of adulterated •.products• (RB 1-

Aug-2016) are recognized:• Sexual Enhancement: This category is also referred to as Change to read:the Erectile Dysfunction (ED) category. It encompasses a

RECOMMENDED ANALYTICAL METHODOLOGIES1.In the United States, dietary supplements are defined as substances that are •

.Adulteration analysis• (RB 1-Aug-2016) may be broadly catego-ingested, in agreement with 21 U.S. Code §321(ff)(2)(A)(i). Definitions ofrized into targeted and nontargeted methods. The distinc-dietary supplements, nutritional supplements, functional foods, and bioactivetion between these types may be subtle, and a minor ad-food additives may vary extensively, depending on local or national legisla-justment to the methodology will transform a nontargetedtion. In the marketplace, there is a trend toward expanding the mode ofmethod into a targeted method.delivery of the adulterating compounds to routes not covered by the regula-

Targetedtory definition for dietary supplements, i.e., topical oils, creams, lotions, e-These techniques are warranted when the analytes arecigarettes, chewing gums, sprays, and others. Such novel delivery systemsknown. An example of a targeted approach would bepresent unique challenges, particularly from the standpoint of sample prepa-monitoring a chromatographic run at a particular wave-ration, and are not considered for the purposes of this chapter to be dietarylength (or mass), and quantifying the analyte that ap-supplements. However, recognizing the emerging threat, USP chooses topears within a predefined retention time window.highlight the existence of these products. In no way should mention ofTargeted analysis is conceptually straightforward, becausethese products be interpreted as a comment on their legal status or beit relies on pre-existing knowledge of the analyte and al-perceived as an expansion of the definition of DS.

2016 The United States Pharmacopeial Convention All Rights Reserved.

C173956_160319-M8206-NBDS2015, Rev. 0 20160527

Revision Bulletin2 ⟨2251⟩ Screening for Undeclared Drugs and Drug Analogues Official August 1, 2016

lows optimization of test methodology for its reliable de- Table 1 (Continued)tection. The targeted approach also is a rarity in the •. Time Solution A Solution Badulterated products analysis,• (RB 1-Aug-2016) where the (min) (%) (%)nature of the analyte may be anticipated only tentatively,

24 95 5and variable amounts of multiple adulterants belonging31 95 5to several functional categories are commonplace.

NontargetedDiluent: Acetonitrile and water (50:50)These methods are better suited to a broad-spectrum de-Standard solution: 100 µg/mL each of USP Sildenafil Cit-tection requirement presented by adulterated •.products.rate RS, USP Tadalafil RS, •.and• (RB 1-Aug-2016) USP• (RB 1-Aug-2016) Nontargeted screening trades precise knowl-Vardenafil Hydrochloride RS in Diluentedge of the analyte identity, along with specificity and

Sample solution: Combine one-fifth of the dosage unit,accuracy, for a wider detection scope. Examples of10–20 mg of bulk material, or a small fragment of thenontargeted chromatographic screening include acquisi-capsule shell (3 mm × 3 mm) with 10 mL of Diluent, soni-tion of photodiode array data and full mass-spectral scan-cate for 30 min, and pass through a 0.2-µm PTFE syringening following a chromatographic separation. The proce-filter.dures in this chapter are written with an eye toward

Chromatographic systemapplying all techniques in a nontargeted mode, even the(See Chromatography ⟨621⟩, System Suitability.)ones considered to be inherently targeted, thereby facili-Mode: LCtating detection of a suspect adulterant even in the ab-Detector: Photodiode array, 200–400 nmsence of a matching reference compound.Analytical wavelength: 290 nmIt is generally recommended to apply a broadlyColumn: 2.1-mm × 15-cm; 5-µm packing L13

. nontargeted methodology first, followed by a targetedColumn temperature: 40°procedure. It is crucial to clearly define the end-purposeFlow rate: 0.2 mL/minof analysis, and only then decide on the appropriate in-Injection volume: 1 µLstrumentation and assemble a logical testing strategy

System suitabilityfrom the procedures provided. Thus, application of aSample: Standard solutionnontargeted screening method may satisfy the require-Suitability requirementsments of a •.• (RB 1-Aug-2016) manufacturer for the purposesColumn efficiency: NLT 3000 theoretical platesof monitoring bulk raw materials. Conversely, a labora-Tailing factor: NMT 1.5tory requiring a higher level of evidence to enact an en-

Analysisforcement action may opt for a two-step procedure: aSample: Sample solutionpreliminary screen, followed by confirmatory analysis ofExamine the UV spectra of the prominent peaks for simi-suspect samples.larity to those in the •.Standard solution• (RB 1-Aug-2016) orUSP Reference Standards recommended for •.• (RB 1-Aug-2016) other known PDE5 inhibitor compounds (Figure 1 andscreening are listed at the end of each relevant Appendix.Table 5). Typical retention times of several PDE5 inhibi-However, considering the rate of propagation of struc-tors are provided in Table 5. However, neither retentiontural analogues and proliferation of newly developed “de-time match nor the absorbance spectrum similaritysigner” molecules, establishing and maintaining an all-in-should be construed as sufficient confirmation of theclusive catalog of reference materials •.• (RB 1-Aug-2016) is •.

chemical identity of an adulterant.both• (RB 1-Aug-2016) challenging and impractical. Several2. HPLC with Mass-Spectrometric Detectioncommercial sources of the compounds of interest exist.2

. •.Preferably,• (RB 1-Aug-2016) a mass-spectrometric detector isPlease note that mention of the external reference mater-connected in sequence to the UV-Vis detector. The set-ials suppliers does not in any way constitute their en-tings below apply to an ion-trap mass spectrometer.dorsement, •.as• (RB 1-Aug-2016) neither does the listing ofOther MS detectors are suitable; however, it is advisablereagents, supplies, and instrumentation. to use spectrometers that possess MS/MS capability.

Solution A: 0.1% Formic acid in waterAPPENDIXSolution B: 0.1% Formic acid in acetonitrileMobile phase: See Table 2.

Change to read:

Table 2• APPENDIX A. SCREENING METHODOLOGIES FOR PDE5 INHIBITORSTime Solution A Solution B1. HPLC with Photodiode Array Detection(min) (%) (%)Solution A: 0.1% Formic acid in water

Solution B: 0.1% Formic acid in acetonitrile 0 95 5Mobile phase: See Table 1. 15 5 95

23 5 95Table 1 24 95 5

Time Solution A Solution B 31 95 5(min) (%) (%)

Diluent: Acetonitrile and water (50:50)0 95 5Standard solution: 5 µg/mL each of USP Sildenafil Cit-

15 5 95 rate RS, USP Tadalafil RS, •.and• (RB 1-Aug-2016) USP23 5 95 Vardenafil Hydrochloride RS in Diluent

Sample solution: Combine one-fifth portion of the dos-2

.CacheSyn (http://www.cachesyn.com/); Santa Cruz Biotechnology, Inc. age unit, 10–20 mg of bulk material, or a small fragment(http://www.scbt.com); TLC Pharmachem (http://www.tlcpharmachem.com/); and Toronto Research Chemicals (http://www.trc-canada.com/) are 3

.The procedure was developed on the Agilent Technologies Zorbax SB-C18some of the potential sources of rare and hard-to-find adulterant reference column.materials.


C173956_160319-M8206-NBDS2015, Rev. 0 20160527


of the capsule shell (3 mm × 3 mm) with 10 mL of Dilu- Application volume: 3 µL, as 8-mm bandsent, sonicate for 30 min, and pass through the 0.2-µm Relative humidity: Condition the plate to a relative hu-PTFE syringe filter. Dilute the filtrate 20-fold with Diluent midity of 47% using a suitable device in the presence ofbefore injection. a saturated solution of potassium isothiocyanate.

Chromatographic system Temperature: Ambient(See Chromatography ⟨621⟩, System Suitability.) Saturation: 20 min, with paperMode: LC Developing distance: 6 cmDetector: UV 290 nm Derivatization reagent: NoneColumn: 2.1-mm × 15-cm; 5-µm packing L14

. Drying: 5 min in a current of cold airColumn temperature: 40° Detection 1: Visual, under illumination with 254- andFlow rate: 0.2 mL/min 365-nm UV lightInjection volume: 1 µL Detection 2: UV-Vis spectrometry (scanning densitome-

Mass spectrometric system5. ter), 190–550 nm

(See Mass Spectrometry ⟨736⟩.) Detection 3: Mass spectrometry, m/z •.90• (RB 1-Aug-

Ionization: ESI 2016)–1050Polarity: Positive or negative Mass spectrometric system7

. Sheath gas: 35 mL/min (See Mass Spectrometry ⟨736⟩.)Sweep gas: 5 mL/min Ionization: ESICapillary temperature: 300° Polarity: Positive, negative, or rapid switchingSource voltage: 5 kV Desolvation gas (N2): 300 L/hCollision: 45 meV Cone gas (N2): 80 L/hScanning: m/z •.90• (RB 1-Aug-2016)–1050 and dependent Temperaturesscan on the most intense ion ESI probe: 105°

System suitability Desolvation: 150°Sample: Standard solution Capillary voltage: 3.0 kVSuitability requirements Cone voltage: 50 VResolution: NLT 2.0 between vardenafil and sildenafil Scanning: m/z •.90• (RB 1-Aug-2016)–1050peaks System suitability

Tailing factor: NMT 1.5 Sample: Standard solutionAnalysis Suitability requirements: Under UV light at 254 nm,Sample: Sample solution sildenafil, tadalafil, and vardenafil appear as dark bandsCompare mass-to-charge ratios of the molecular ions against the fluorescent background. Under UV light at[M+H]+

. or [M−H]−. and fragments to those of the Stan- 365 nm, sildenafil, tadalafil, and vardenafil appear as

dard solution or other known analytes listed in Table 4. blue fluorescent bands.Typical retention times of several common PDE5 inhibi- Analysis: Inspect the plate under short-wave (254 nm)tors are provided in Table 5. and long-wave (365 nm) UV light. PDE5 inhibitors appear

3. High Performance Thin-Layer Chromatography as dark bands against the fluorescent background at 254(HPTLC) with Visual, UV, and/or MS Detection nm and typically exhibit different shades of blue fluores-Standard solution: A composite of 0.2-mg/mL each of cence •.under• (RB 1-Aug-2016) 365 nm. Note the similaritiesUSP Sildenafil Citrate RS, USP Tadalafil RS, and USP in RF values between the bands in the Standard solutionVardenafil Hydrochloride RS in methanol, with sonication and Sample solution; these may be informative, •.how-if necessary. Additional reference materials may be availa- ever they do not constitute sufficient proof of identity.ble commercially. • (RB 1-Aug-2016) Relative intensities of the bands permit ap-

Sample solution: Comminute 1 dosage unit, including proximation of the amounts. Using scanning densitome-the capsule shell and tablet coating, or about 500 mg of try, obtain UV spectra of the prominent bands in theraw material; combine with 10 mL of methanol, and sub- Sample solution, and compare them to those of the PDE5ject to ultrasonication for 30 min. Centrifuge or filter the inhibitors in the •.Standard solution• (RB 1-Aug-2016) andsolution, and use the supernatant. [NOTE—Upon develop- those provided in Table 5 and Figure 1. Mass-spectromet-ment, if the chromatographic bands appear too saturated ric interface, if available, may facilitate more definitive as-and UV densitometric spectra are distorted, dilute the signment of the analyte bands: compare mass-to-chargeSample solution 10-fold with methanol.] ratios of the molecular ions [M+H]+

. or [M−H]−. and frag-

Developing solvent system: tert-Butyl methyl ether, ments to those of the common adulterants listed in Tablemethanol, and 28.0% (w/w) ammonium hydroxide 4.(20:2:1). [NOTE—Strength of ammonium hydroxide was 4. Ambient Ionization Mass Spectrometryfound to be crucial for adequate method performance. It Diluent: Acetonitrile and water (50:50), with 0.1% formicis therefore advisable to establish the titer of higher-con- acidcentration ammonia6

. and to adjust the latter to exactly Standard solution: A composite solution containing28.0% immediately before the experiment.] 20 µg/mL each of USP Sildenafil Citrate RS, USP Tadalafil

Chromatographic system RS, and USP Vardenafil Hydrochloride RS in Diluent. Addi-(See HPTLC for Articles of Botanical Origin ⟨203⟩.) tional reference materials may be available commercially.•

.• (RB 1-Aug-2016) Sample solution: Grind the entire dosage form, includ-Adsorbent: Chromatographic silica gel with an average ing the capsule shell and tablet coating, to a fine pow-particle size of 5 µm der. Weigh about 50 mg of the resulting powder, or

about 50 mg of bulk material, and combine with 5 mL of4

.The procedure was developed on the Agilent Technologies Zorbax SB-C18column. 7

.Procedure was developed using Expression CMS mass spectrometer from5

.The settings were found appropriate for ThermoElectron LTQ XL Linear Ion Advion, equipped with a TLC-MS interface available from CAMAG. If otherTrap Mass Spectrometer. Users will need to optimize their respective instru- mass spectrometers are used, relevant settings will have to be optimized.mentation according to the manufacturer’s recommendations. The bands were directly eluted with a mixture of water and acetonitrile6

.32% Ammonia solution is available from EMD Millipore. (80:20) containing 0.1% formic acid.


C173956_160319-M8206-NBDS2015, Rev. 0 20160527


Diluent. Cap tightly, subject to ultrasonication for 2 min, according to the recommendations of the equipmentand vortex thoroughly. Centrifuge or filter the resulting manufacturer.solution, and dilute an aliquot of the supernatant or fil-trate 100-fold with Diluent. Table 3

Mass spectrometric systemParameter 1

.H-NMR Qualitative Measurement(See Applications of Mass Spectrometry ⟨1736⟩, Mass Spec-Pulse program Single pulse 1

.Htrometers, Ionization Procedures, Ambient Ionization Proce-dures.) Spectral width 14 ppm (−1 to 13 ppm)

Ionization: Ambient with thermal desorption Transmitter offset Center of spectral widthMode: Thermal profile Relaxation delay 5–10 sPolarity: Positive, negative, or rapid switching

Acquisition time 2–5 sGas temperature: 150°, 250°, 350°, and 450°Number of data pointsScanning: m/z •.90• (RB 1-Aug-2016)–1050per FIDa

. NLT 16,000System suitability: Deposit 3-µL aliquots of the StandardTemperature 25°solution onto the disposable sample cards. Set the com-

patible mass spectrometer to a 30-s acquisition of •.

a.Free induction decay.

90• (RB 1-Aug-2016)–1050 Da. Using one sample card for eachSystem suitability: Acquire a 1

.H spectrum of the •.Stan-temperature setting, acquire mass spectra at each of thedard solution• (RB 1-Aug-2016) using the settings outlined infollowing desorption gas temperatures: 150°, 250°, 350°,Data collection. Record a sufficient number of scans toand 450° in positive ionization mode. Switch polarity,ensure that signal-to-noise ratio of the TMS signal is NLTand re-acquire spectra at the same four temperatures in10.the negative ionization mode. [NOTE—If the mass spec-

Analysis: Acquire a 1.H spectrum of the Sample solutiontrometer permits rapid polarity switching, both positive

under the conditions outlined in Data collection. Record aand negative spectra may be acquired simultaneously us-sufficient number of scans to ensure that the signal-to-ing a single sample.] Confirm that the [M+H]+

. or [M−H]−.

noise ratio of the TMS signal is NLT 10. Reference allions of sildenafil, tadalafil, and vardenafil are observed asacquired spectra to the 1

.H signal of TMS (0 ppm). Meas-listed in Table 4.ure and record the chemical shift and multiplicity of theAnalysis: Deposit 3-µL aliquots of the Sample solutionNMR signals in the spectra of the Standard solutions andonto the disposable sample cards, and follow the proce-Sample solution. Compare the 1

.H NMR spectrum of thedure outlined above for the Standard solution. In theSample solution to those of the Standard solutions, payingevent that a single ion dominates the mass spectrum atparticular attention to the aromatic region (5–9 ppm).every temperature setting, dilute the Sample solutionsDetermine whether the chemical shift and multiplicity of10-fold with Diluent and re-analyze. Compare mass-to-the NMR signals in the Sample solution exhibit sufficientcharge ratios of the molecular ions [M+H]+

. or [M−H]−. and

similarity to those found in the Standard solutions.fragments to those of the known analytes listed in Table6. Bioassay8

. 4.PDE5 enzyme9

. stock solution: Prepare a concentration5. NMR Spectroscopy–Low-Field and High-Fieldof approximately 3000 Units/µL. If necessary, dilute with(See Nuclear Magnetic Resonance Spectroscopy ⟨761⟩, Quali-40 mM tris(hydroxymethyl)aminomethane hydrochloridetative and Quantitative NMR Analysis.)(tris-HCl), pH 8.0, 110 mM sodium chloride (NaCl), 2.2[NOTE—Deuterated acetonitrile (CD3CN) should be NLTmM potassium chloride (KCl), 3 mM dithiothreitol, and99.8 atom % D, and should contain 0.05% tetramethylsi-20% glycerin. Vortex gently to mix.lane (TMS) as a chemical shift reference. Use of solvents

PDE5 working solution (100 Units per 6.5 µL): Combinein sealed ampules is recommended. NMR tubes should400 µL of PDE-Glo Reaction Buffer 5X, 10 µL of PDE5be suitable for use at the selected magnetic fieldenzyme stock solution, and 1590 µL of •.• (RB 1-Aug-2016)strength.]water. Vortex gently to mix.Standard solutions: Dissolve 10 mg of USP Sildenafil Cit-

cGMP solution: Combine 400 µL of PDE-Glo Reactionrate RS, USP Tadalafil RS, or USP Vardenafil HydrochlorideBuffer 5X, 40 µL of 1-mM cGMP stock solution, andRS in separate 1-mL aliquots of CD3CN, and transfer1560 µL of •.• (RB 1-Aug-2016) water. Mix thoroughly by700-µL aliquots of the resulting solutions into individualvortexing.NMR tubes. Additional reference materials are available

commercially. 8.The procedures were developed using commercial Promega PDE-Glo

Sample solution: Grind the entire dosage form, includ- Phosphodiesterase Assay Kit, Catalog # V1361. It includes the following re-ing the capsule shell and tablet coating, to a fine pow- agents: PDE-Glo Reaction Buffer 5X (Catalog # V133A); PDE-Glo Detec-der. Transfer 100–200 mg of the ground material, or an tion Buffer 5X (Catalog # V134A); Protein Kinase A Solution (Catalog #equivalent amount of bulk raw material powder, into a V135A); PDE-Glo Termination Buffer 5X (Catalog # V136A); cGMP Stock5-mL sealable glass vial. Add 1 mL of CD3CN, vortex Solution, 1 mM (Catalog # V641A); cAMP, 1 mM (Catalog # V642B); Kin-thoroughly, and allow the solids to settle. Transfer about ase-Glo Substrate (Catalog # V672A); and Kinase-Glo Buffer (Catalog #700 µL of the supernatant to an NMR tube, taking care V673A). Kits from alternative suppliers may also be used, e.g., BPS Science,to minimize transfer of solids. Catalog # 60350, although re-optimization of test procedures will be re-

Instrument performance qualification quired.(See Nuclear Magnetic Resonance Spectroscopy ⟨761⟩.) 9

.The procedures were developed using human phosphodiesterase 5A fromMagnetic field strength: NLT 42.5 MHz (1

.H operating BPS Bioscience Catalog # 60050. The enzyme is available from numerousfrequency) suppliers, e.g., Sigma-Aldrich Catalog # E9034.

Data collection: Use the parameters specified in Table 3;perform 90° pulse width calibration before the analysis


C173956_160319-M8206-NBDS2015, Rev. 0 20160527


100-mM IBMX stock solution in DMSO: Prepare a 22.2- tion with 240 µL of Reaction buffer, and mix thoroughlymg/mL solution of 3-isobutyl-1-methylxanthine (IBMX) in by vortexing; •.the last dilution is the Sample solution.dimethylsulfoxide (DMSO), e.g., dissolve 100 mg of IBMX • (RB 1-Aug-2016)

in 4.5 mL of DMSO. Mix thoroughly by vortexing. AnalysisReaction buffer: Combine 400 µL of PDE-Glo Reaction 1. Dispense 12.5-µL aliquots of the Standard solution,Buffer 5X and 1600 µL of •.• (RB 1-Aug-2016) water. Mix thor- Control solution, and Sample solution into microplateoughly by vortexing. wells, in triplicate. Use a white, flat-bottom, opaque

Reaction buffer with 4% DMSO: Combine 400 µL of polystyrene, nontreated, 96-well microtiter plate.10.

PDE-Glo Reaction Buffer 5X, 80 µL of DMSO and [NOTE—Do not use treated plates, black plates, or1520 µL of •.• (RB 1-Aug-2016) water. Mix thoroughly by clear plates.] vortexing. 2. Add 6.5 µL of PDE5 working solution to each well.

Termination buffer: Combine 400 µL of PDE-Glo Ter- Incubate for 5 min.mination Buffer 5X, 40 µL of 100-mM IBMX stock solution 3. Add 6.0 µL of cGMP solution to effect a 5-µMin DMSO, and 1560 µL of •.• (RB 1-Aug-2016) water. Mix thor- cGMP concentration in a 25-µL volume. Incubateoughly by vortexing. for 30 min.

Detection buffer: Combine 400 µL of PDE-Glo Detec- 4. Add 12.5 µL of Termination buffer. Incubate for 5tion Buffer 5X, 16 µL of Protein Kinase A Solution, and min.1584 µL of •.• (RB 1-Aug-2016) water. Vortex gently to mix. 5. Add 12.5 µL of Detection buffer. Incubate for 20

Kinase-Glo reagent: Add 10 mL of Kinase-Glo Buffer min.to the vial of Kinase-Glo Substrate, and vortex gently. 6. Add 50 µL of Kinase-Glo reagent. Incubate for 10

Standard solution (400 nM): Dissolve 5 mg of USP min.Sildenafil Citrate RS in 3.0 mL of DMSO to obtain a 2.5- 7. Record luminescence at 560 nm with a microplatemM stock solution. Combine a 10-µL aliquot of the re- luminometer at 0.5 s/well.sulting solution with 240 µL of DMSO, and mix thor- [NOTE—Incubate the plate at room temperature, prefera-oughly (100 µM). Combine a 10-µL aliquot of the result- bly using a plate shaker.]ing solution with 90 µL of DMSO, and mix thoroughly Calculate average luminescence values for the replicate(10 µM). Combine a 10-µL aliquot of the resulting solu- preparations. Assess the degree of PDE5 inhibition in thetion with 240 µL of Reaction buffer, and mix thoroughly Sample solution relative to that observed in the Standard(400 nM); •.the last dilution is the Standard solution. solution and Control solution. Inhibition of PDE5 is mani-• (RB 1-Aug-2016) fested as reduction of luminescence: the samples that

Control solution: Combine 10 µL of DMSO with 240 µL exhibit suppression of luminescent output comparableof Reaction buffer, and mix thoroughly by vortexing. to or in excess of that observed with the Standard solu-

Sample solution: Grind the entire dosage form, includ- tion are likely adulterated with synthetic PDE5 inhibitors.ing the capsule shell and tablet coating, to a fine pow- • USP REFERENCE STANDARDS ⟨11⟩der. Transfer 100 mg of the ground material into a 5-mL USP Sildenafil Citrate RSpolypropylene vial. Add 3.0 mL of DMSO, and vortex for USP Tadalafil RS60 s. Allow solids to settle, combine 50 µL of the clear USP Vardenafil Hydrochloride RSsupernatant with 200 µL of DMSO, and mix thoroughly

10.Conforming plates are available from Corning (Costar 3912 or Costarby vortexing. Combine a 10-µL aliquot of the resulting

3963), Thermo Scientific (Nunc Catalog #236105), and other vendors.solution with 90 µL of DMSO, and mix thoroughly byvortexing. Combine a 10-µL aliquot of the resulting solu-

Table 4. Mass Spectral Data for Select PDE5 Inhibitorsa.

Chemical# Name CAS Number Formula Exact Mass [M+H]+

. [M−H]−. Fragments

455 [M+Na]+.,

— 391, 311, 269,1 Acetaminotadalafil 1446144-71-3 C23H20N4O5 432.1434 433 250

329, 300, 285,— 268, 256, 242,•

.147676-78-6• (RB 1-Aug-

2 Acetil acid 2016) C18H20N4O4 356.1485 357 166, 131449, 439, 420,404, 396, 381,355, 353, 339,

—325, 324, 311,

Acetildenafil 297, 285, 166,3 (Hongdenafil) 831217-01-7 C25H34N6O3 466.26924 467.28 127, 111, 99, 97

439, 396, 341,—

4 Acetylvardenafil 1261351-28-3 C25H34N6O3 466.2692 467 317, 270432, 377, 313,Aildenafil

— 311, 283, 113,(Dimethyl-489.23 99sildenafil,

— 487.40 460, 310, 2825 Methisosildenafil) 496835-35-9 C23H32N6O4S 488.22057a

.Compiled from peer-reviewed literature, and communications with USP collaborators. See corresponding chemical structures in Figure 2.b

.Bold, fragment subjected to MS3. fragmentation.

c.Italics, MS3

. fragments derived from the parent fragment (bold).


C173956_160319-M8206-NBDS2015, Rev. 0 20160527


Table 4. Mass Spectral Data for Select PDE5 Inhibitorsa. (Continued)



269, 262, 241,— 239, 224, 197,

391.14 169362, 298, 262,

—389.1248 234, 233, 2326 Aminotadalafil 385769-84-6 C21H18N4O4 390.1328

(S,R)-Aminotadalafil[(+)-trans- —•

.1093940-70-5• (RB 1-

7 Aminotadalafil] Aug-2016) C21H18N4O4 390.1328 391 No data8 Avanafil 330784-47-9 C23H26N7O3Cl 483.1786 484.186 — 375, 349, 221

•.1446089-82-2• (RB 1- —9 Benzylsildenafil Aug-2016) C28H34N6O4S 550.2362 551 377, 283

C25H25N3O4 431.18451 432.25 310,b. 282,c. 197,—

10 N-Butylnortadalafil 171596-31-9 169Carbodenafil

—11 (Fondenafil) 944241-52-5 C24H32N6O3 452.2536 453 283

360, 311, 291,—

12 Chlorodenafil 1058653-74-9 C19H21ClN4O3 388.8485 390 254, 183, 136429, 395, 349,334, 302, 287,

—262, 229, 159,

13 Chloropretadalafil 171489-59-1 C22H19ClN2O5 426.0982 427 135488, 354, 297,

— 283, 215, 166,•.1446089-83-3 • (RB 1-

14 Cinnamyldenafil Aug-2016) C32H38N6O3 554.3005 554 117, 9115 Cyclopentynafil 1173706-34-7 C26H36N6O4S 528.2519 529 — 461, 153

381, 351, 327,—

409 299, 285, 272Depiperazinothi- •.1353018-10-6• (RB 1-

— 407.0845 378, 31416 osildenafil Aug-2016) C17H20N4O4S2 408.0926418, 377, 360,

— 311, 299, 283,17 Descarbonsildenafil 1393816-99-3 C21H30N6O4S 462.2049 463 255, 151, 87

392, 377, 376,329, 313, 312,

—N- 299, 284, 283,

18 Desethylvardenafil 448184-46-1 C21H28N6O4S 460.18927 461.20 151Desmethyl- •

.147676-79-7• (RB 1-Aug- •.438.523• (RB 1- —

2016)19 carbodenafil C23H31N6O3 Aug-2016) 439.2451 339, 311N-Desmethyl- 377, 313, 311,

—20 sildenafil 139755-82-1 C21H28N6O4S 460.18927 461.19 299, 283

Dimethylace- •.1417999-76-8• (RB 1- —

21 tildenafil Aug-2016) C25H34O3N6 466.2771 467 279, 149, 177•

.1609405-33-5• (RB 1- —22 Dioxo-acetildenafil Aug-2016) C25H30N6O5 494.2278 495 No dataDithio-desmethyl- 1333233-46-7 C23H30N6OS2 470.6572

—23 carbodenafil 471.1991 371, 343

327, 298, 285,—

24 Gendenafil 147676-66-2 C19H22N4O3 354.16919 355.31 283, 256, 24225 Gisadenafil 334826-98-1 C23H33N7O5S 519.22639 520 — No data

467, 461, 377,313, 311, 283,

—127, 111, 113,

26 Homosildenafil 642928-07-2 C23H32N6O4S 488.22057 489.23 99, 97Hydroxyace-tildenafil 465, 447, 439,

—(Hydrox- 396, 353, 339,

27 yhongdenafil) 147676-56-0 C25H34N6O4 482.26415 483.27 325, 311, 297Hydrox-

—28 ychlorodenafil 1391054-00-4 C19H23ClN4O3 390.1459 391 No dataa



c.Italics, MS3



C173956_160319-M8206-NBDS2015, Rev. 0 20160527





487, 461, 423,377, 312, 311,

— 283, 284, 225,166, 129, 112,

505.22 99, 97Hydrox-— 503.31 475, 310, 28229 yhomosildenafil 139755-85-4 C23H32N6O5S 504.21549

2-Hydrox- 312, 284, 197,—

30 ypropylnortadalafil 1353020-85-5 C24H23N3O5 433.16377 434.15 169Hydroxythi-ohomosildenafil(Hydrox-yhomosildenafil — 503, 477, 461,thione, 419, 393, 355,Sulfohydrox- 354, 327, 325,

31 yhomosildenafil) 479073-82-0 C23H32N6O4S2 520.19264 521.20 291Hydroxythi- •

.No data• (RB 1-Aug-—32 ovardenafil 912576-30-8 C23H32N6O4S2 520.1926 521 2016)

•.505.26 •

.344, 312, 253,• (RB — 169, 99• (RB 1-

1-Aug-2016) Aug-2016)

•.503• (RB 1-—

Aug-2016) 475, 310, 28233 Hydroxyvardenafil 224785-98-2 C23H32N6O5S 504.2155Imidazosagatriazi-none — •

.• (RB 1-Aug-2016) 284,34 (Desulfovardenafil) 139756-21-1 C17H20N4O2 312.1586 313 256, 169, 151

483 — —453, 422, 379,

— 336, 325, 311,•.1335201-06-3• (RB 1-

481 30935 Isopiperazinonafil Aug-2016) C25H34N6O4 482.2642Lodenafil carbon- 518, 487, 377,

—36 ate 398507-55-6 C47H62N12O11S2 1034.4102 1035 311

488, 404, 362,—

37 Mirodenafil 862189-95-5 C26H37N5O5S 531.6698 532 296, 268489, 377, 142,

—38 Mutaprodenafil 1387577-30-1 C27H35N9O5S2 629.7635 630.2279 113

307, 289, 261,— 217, 176, 154,

39 Nitrodenafil 147676-99-1 C17H19N5O4 357.3647 358 136, 107, 8940 Nitroso-prodenafil 1266755-08-1 C27H35N9O5S2 629.2203 630 — 142

425, 406, 396,380, 367, 355,

— 353, 339, 325,Noracetildenafil 324, 313, 297,

41 (Demethylhongdenafil) 949091-38-7 C24H32N6O3 452.25359 453.26 296, 253Norneosildenafil(Piperidino — 432, 377, 329,

42 sildenafil) 371959-09-0 C22H29N5O4S 459.19403 460.20 311, 299, 283329, 307, 289,

— 176, 154, 136,43 Norneovardenafil 358390-39-3 C18H20N4O4 356.1485 357 107, 99

Nortadalafil 262, 234, 233,44 (Demethyltadalafil) 171596-36-4 C21H17N3O4 375.1219 376 374.1138 232

N-Octylnortadalafil—

45 (Octylnortadalafil) 1173706-35-8 C29H33N3O4 487.2471 488 366, 227451, 396, 354,

— 339, 312, 297,•.1446144-70-2• (RB 1-

46 Oxohongdenafil Aug-2016) C25H32N6O4 480.2485 481 289483 — —Piperazinonafil

453, 435, 348,(Piperazonifil,— 336, 321, 311,Dihydroace-

481 309, 282, 26747 tildenafil) 1335201-04-1 C25H34N6O4 482.2642a



c.Italics, MS3



C173956_160319-M8206-NBDS2015, Rev. 0 20160527





Piperidino ace-tildenafil 410, 408, 355,

—(Piperiace- 353, 341, 325,

48 tildenafil) 147676-50-4 C24H31N5O3 437.2427 438 297, 288432, 403, 391,377, 349, 329,

Piperidi- 312, 311, 301,—

novardenafil 299, 284, 283,(Piperidenafil, 270, 256, 169,

49 Pseudovardenafil) 224788-34-5 C22H29N5O4S 459.19403 460.20 151Propoxyphenyl •

.1391053-82-9• (RB 1- —50 aildenafil Aug-2016) C24H34N6O4S 502.2362 503 252

Propoxyphenylhydrox- 501, 475, 391,yhomosildenafil — 331, 325, 299,(Methylhydrox- 283, 129, 112,

51 yhomosildenafil) 139755-87-6 C24H34N6O5S 518.2311 519 99Propoxyphenyl 447, 391, 325,

—52 sildenafil 877777-10-1 C23H32N6O4S 488.2205 489.2272 299, 283, 100

Propoxyphenylthioaildenafil(Propoxyphenyl —thi-

53 omethisosildenafil) 856190-49-3 C24H34N6O3S2 518.2133 519 260Propoxyphenyl 517, 359, 341,thiohydrox- — 315, 299, 271,

54 yhomosildenafil 479073-90-0 C24H34N6O4S2 534.2083 535.2150 129, 112, 99447, 418, 391,377, 374, 346,

— 329, 311, 297,283, 255, 163,

475.21 160, 100— 473.45 445, 310, 28255 Sildenafil 139755-83-2 C22H30N6O4S 474.20492

302, 268, 262,— 250, 240, 197,

390 169, 135262, 234, 233,

—388.1288 23256 Tadalafil (Tildenafil) 171596-29-5 C22H19N3O4 389.13756

(−)-trans-Tadalafil 779 [2M+H]+.,—

57 (ent-Tadalafil) 629652-72-8 C22H19N3O4 389.1376 390 262, 250, 135

Thioaildenafil(Sulfoaildenafil,Thi-omethisosildenafil,

—Sulfodimethylsildenafil,Dimethylthi- 448, 393, 327,

58 osildenafil) 856190-47-1 C23H32N6O3S2 504.19773 505.21 299, 113, 99Thiohomosildenafil 477, 421, 393,(Sulfohomosildenafil, 357, 355, 343,

—Homosildenafil 327, 315, 299,

59 thione) 479073-80-8 C23H32N6O3S2 504.19773 505.21 271, 113, 99Thioquinapiperifil 363, 246, 225,

—60 (KF31327) 220060-39-9 C24H28N6OS 448.2045 449 204, 121

407, 393, 343,Thiosildenafil 341, 327, 315,

—(Sulfosildenafil, 313, 299, 283,

61 Sildenafil thione) 479073-79-5 C22H30N6O3S2 490.18208 491.19 271, 163, 99474, 418, 347,

—62 Udenafil 268203-93-6 C25H36N6O4S 516.2519 517.260 325, 299, 283a



c.Italics, MS3



C173956_160319-M8206-NBDS2015, Rev. 0 20160527





461, 420, 377,376, 375, 346,339, 329, 312,

—299, 284, 283,169, 151, 123,

489.2274 99— 487.33 459, 310, 28263 Vardenafil 224785-90-4 C23H32N6O4S 488.22057

Xanthoanthrafil 344, 252, 223,—

64 (Benzamidenafil) 1020251-53-9 C19H23N3O6 389.15869 390.31 151, 107, 91a



c.Italics, MS3


Table 5. UV Absorbance Maxima and Retention Time Data for Select PDE5 Inhibitorsa.

RelativeUV Ab- Retention

UV Ab- sorbance Timesorbance Spectrum Retention with Re-

Chemical Maxima Type Time spect to# Name CAS Number Formula (nm) (Figure 1) (min)b

. Sildenafil202, 222,

1 Acetaminotadalafil 1446144-71-3 C23H20N4O5 282 b 14.3 1.1230, 260,•

.147676-78-6 • (RB 1-Aug- — — —2 Acetil acid 2016) C18H20N4O4 2853 Acetildenafil (Hongdenafil) 831217-01-7 C25H34N6O3 234, 282 e 12.5 1.0

218, 246,4 Acetylvardenafil 1261351-28-3 C25H34N6O3 268(s)c

. d 11.4 0.9Aildenafil (Dimethyl-sildenafil,

5 Methisosildenafil) 496835-35-9 C23H32N6O4S 226, 294 a 13.3 1.0200, 220,

284,6 Aminotadalafil 385769-84-6 C21H18N4O4 290(s) b 14.2 1.1

(S,R)-Aminotadalafil [(+)-trans- — — —•

.1093940-70-5• (RB 1-

7 Aminotadalafil] Aug-2016) C21H18N4O4 225, 2838 Avanafil 330784-47-9 C23H26N7O3Cl 198, 244 f 13.0 1.09 •

.1446089-82-2• (RB 1- C28H34N6O4S 291 — — —Benzylsildenafil Aug-2016)

10 N-Butylnortadalafil 171596-31-9 C25H25N3O4 222, 284 — — —11 Carbodenafil (Fondenafil) 944241-52-5 C24H32N6O3 295 — — —

211, 235,— — —

12 Chlorodenafil 1058653-74-9 C19H21ClN4O3 279204, 222,

13 Chloropretadalafil 171489-59-1 C22H19ClN2O5 284 b 17.4 1.314 Cinnamyldenafil •

.1446089-83-3• (RB 1- C32H38N6O3 239 — — —Aug-2016)

15 Cyclopentynafil 1173706-34-7 C26H36N6O4S 218, 290 — — —16 Depiperazinothiosildenafil •

.1353018-10-6• (RB 1- C17H20N4O4S2 295, 354 — — —Aug-2016)

17 Descarbonsildenafil 1393816-99-3 C21H30N6O4S 225, 295 — — —18 N-Desethylvardenafil 448184-46-1 C21H28N6O4S 226, 246(s) d 11.9 0.919 Desmethylcarbodenafil •

.147676-79-7• (RB 1- C23H31N6O3 226, 296Aug-2016) a 11.9 0.9

20 N-Desmethylsildenafil 139755-82-1 C21H28N6O4S 224, 294 a 12.9 1.021 Dimethylacetildenafil •

.1417999-76-8• (RB 1- C25H34O3N6 233, 276 — — —Aug-2016)

a.Compiled from peer-reviewed literature, and contributed by the USP collaborators. See corresponding chemical structures in Figure 2.

b.Retention times derived from the experiments conducted as described in HPLC with Photodiode Array Detection and in HPLC with Mass-Spectrometric

Detection methods.c

.(s) denotes a shoulder.


C173956_160319-M8206-NBDS2015, Rev. 0 20160527


Table 5. UV Absorbance Maxima and Retention Time Data for Select PDE5 Inhibitorsa. (Continued)




. Sildenafil22 Dioxo-acetildenafil •

.1609405-33-5• (RB 1- C25H30N6O5 No data — — —Aug-2016)

Dithio-desmethyl- 258, 285,— — —

23 carbodenafil 1333233-46-7 C23H30N6OS2 35624 Gendenafil 147676-66-2 C19H22N4O3 232, 274 f 16.8 1.325 Gisadenafil 334826-98-1 C23H33N7O5S No data — — —26 Homosildenafil 642928-07-2 C23H32N6O4S 226, 292 a 13.3 1.0

Hydroxyacetildenafil27 (Hydroxyhongdenafil) 147676-56-0 C25H34N6O4 234, 280 e 12.2 0.928 Hydroxychlorodenafil 1391054-00-4 C19H23ClN4O3 212, 303 — — —29 Hydroxyhomosildenafil 139755-85-4 C23H32N6O5S 226, 296 a 12.9 1.030 2-Hydroxypropylnortadalafil 1353020-85-5 C24H23N3O5 222, 284 — — —

Hydroxythiohomosildenafil(Hydroxyhomosildenafilthione,Sulfohydrox- 228, 296,

31 yhomosildenafil) 479073-82-0 C23H32N6O4S2 352 c 15.0 1.2203, 235,

— — —32 Hydroxythiovardenafil 912576-30-8 C23H32N6O4S2 31633 Hydroxyvardenafil 224785-98-2 C23H32N6O5S 216 — — —

Imidazosagatriazinone— — —

34 (Desulfovardenafil) 139756-21-1 C17H20N4O2 212, 25335 Isopiperazinonafil •

.1335201-06-3• (RB 1- C25H34N6O4 221, 290 — — —Aug-2016)

36 Lodenafil carbonate 398507-55-6 C47H62N12O11S2 226, 296 a 15.4 1.237 Mirodenafil 862189-95-5 C26H37N5O5S 216, 248 f 14.5 1.1

218, 240,283, 297, — — —

38 Mutaprodenafil 1387577-30-1 C27H35N9O5S2 33539 Nitrodenafil 147676-99-1 C17H19N5O4 212, 298 — — —40 Nitroso-prodenafil 1266755-08-1 C27H35N9O5S2 241, 301 — — —

Noracetildenafil41 (Demethylhongdenafil) 949091-38-7 C24H32N6O3 234, 280 e 12.3 0.9

Norneosildenafil42 (Piperidino sildenafil) 371959-09-0 C22H29N5O4S 226, 300 a 18.7 1.443 Norneovardenafil 358390-39-3 C18H20N4O4 215, 241 — — —

Nortadalafil— — —

44 (Demethyltadalafil) 171596-36-4 C21H17N3O4 No dataN-Octylnortadalafil

— — —45 (Octylnortadalafil) 1173706-35-8 C29H33N3O4 28146 Oxohongdenafil •

.1446144-70-2• (RB 1-Aug- C25H32N6O4 481 — — —2016)

Piperazinonafil (Piper-azonifil, — — —

47 Dihydroacetildenafil) 1335201-04-1 C25H34N6O4 221, 290Piperidino acetildenafil

48 (Piperiacetildenafil) 147676-50-4 C24H31N5O3 234, 284 e 13.1 1.0Piperidinovardenafil(Piperidenafil,

49 Pseudovardenafil) 224788-34-5 C22H29N5O4S 224, 246(s) d 16.5 1.3215, 225,•

.1391053-82-9• (RB 1- — — —50 Propoxyphenyl aildenafil Aug-2016) C24H34N6O4S 295a

.Compiled from peer-reviewed literature, and contributed by the USP collaborators. See corresponding chemical structures in Figure 2.b

.Retention times derived from the experiments conducted as described in HPLC with Photodiode Array Detection and in HPLC with Mass-SpectrometricDetection methods.c



C173956_160319-M8206-NBDS2015, Rev. 0 20160527


Table 5. UV Absorbance Maxima and Retention Time Data for Select PDE5 Inhibitorsa. (Continued)




. SildenafilPropoxyphenylhydroxyhomosildenafil(Methylhydrox-

51 yhomosildenafil) 139755-87-6 C24H34N6O5S 226, 294 a 13.4 1.052 Propoxyphenyl sildenafil 877777-10-1 C23H32N6O4S 226, 292 a 13.6 1.0

Propoxyphenylthioaildenafil

— — —(Propoxyphenyl 227, 295,

53 thiomethisosildenafil) 856190-49-3 C24H34N6O3S2 355Propoxyphenyl 227, 295,

— — —54 thiohydroxyhomosildenafil 479073-90-0 C24H34N6O4S2 35355 Sildenafil 139755-83-2 C22H30N6O4S 224, 294 a 13.0 1.0

200, 222,284,

56 Tadalafil (Tildenafil) 171596-29-5 C22H19N3O4 292(s) b 15.0 1.2(−)-trans-Tadalafil (ent- 231, 282,

— — —57 Tadalafil) 629652-72-8 C22H19N3O4 289

Thioaildenafil(Sulfoaildenafil, 228,Thiomethisosildenafil, 250(s),Sulfodimethyl sildenafil, 296, 352,

58 Dimethylthiosildenafil) 856190-47-1 C23H32N6O3S2 366(s) c 15.5 1.2228,

Thiohomosildenafil 248(s),(Sulfohomosildenafil, 296, 354,

59 Homosildenafil thione) 479073-80-8 C23H32N6O3S2 370(s) c 14.4 1.1Thioquinapiperifil 211, 268,

— — —60 (KF31327) 220060-39-9 C24H28N6OS 363

228,Thiosildenafil 250(s),(Sulfosildenafil, 296, 356,

61 Sildenafil thione) 479073-79-5 C22H30N6O3S2 368(s) c 15.2 1.262 Udenafil 268203-93-6 C25H36N6O4S 228, 298 f 13.6 1.063 Vardenafil 224785-90-4 C23H32N6O4S 226, 252(s) d 12.1 0.9

Xanthoanthrafil 202, 228,64 (Benzamidenafil) 1020251-53-9 C19H23N3O6 278, 390 f 15.3 1.2a

.Compiled from peer-reviewed literature, and contributed by the USP collaborators. See corresponding chemical structures in Figure 2.b

.Retention times derived from the experiments conducted as described in HPLC with Photodiode Array Detection and in HPLC with Mass-SpectrometricDetection methods.c



C173956_160319-M8206-NBDS2015, Rev. 0 20160527


Figure 1. UV absorbance spectra of select PDE5 inhibitors.11.

11.Data contributed by USP collaborators.


C173956_160319-M8206-NBDS2015, Rev. 0 20160527


Figure 2. Chemical structures of select PDE5 inhibitors.12.

Acetil acid

Acetaminotadalafil

AcetildenafilAcetylvardenafil

AildenafilAminotadalafil

Avanafil(S,R)-Aminotadalafil

Benzylsildenafil

N-Butylnortadalafil

ChlorodenafilCarbodenafil


C173956_160319-M8206-NBDS2015, Rev. 0 20160527


Figure 2. Chemical structures of select PDE5 inhibitors.12. (Continued)

Cinnamyldenafil

Chloropretadalafil

Depiperazinothiosildenafil

Cyclopentynafil

N-DesethylvardenafilDescarbosildenafil

N-DesmethylsildenafilDesmethylcarbodenafil

DioxoacetildenafilDimethylacetildenafil

GendenafilDithio-desmethylcarbodenafil


C173956_160319-M8206-NBDS2015, Rev. 0 20160527



Gisadenafil

Homosildenafil

HydroxyacetildenafilHydroxychlorodenafil

Hydroxyhomosildenafil2-Hydroxypropylnortadalafil

Hydroxythiovardenafil

Hydroxythiohomosildenafil

HydroxyvardenafilImidazosagatriazinone

Isopiperazinonafil

Lodenafil carbonate


C173956_160319-M8206-NBDS2015, Rev. 0 20160527



Mirodenafil

Mutaprodenafil

Nitrodenafil

Nitroso-prodenafil

NorneosildenafilNoracetildenafil

Norneovardenafil

Nortadalafil

Oxohongdenafil

N-Octylnortadalafil


C173956_160319-M8206-NBDS2015, Rev. 0 20160527



PiperazinonafilPiperidino acetildenafil

Piperidinovardenafil

Propoxyphenyl aildenafil

Propoxyphenyl sildenafil

Propoxyphenyl hydroxyhomosildenafil

Propoxyphenyl thioaildenafil

Propoxyphenyl thiohydroxyhomosildenafil

Sildenafil

Tadalafil

Thioaildenafil(−)-trans-Tadalafil


C173956_160319-M8206-NBDS2015, Rev. 0 20160527



Thiohomosildenafil

Thioquinapiperifil

ThiosildenafilUdenafil

Vardenafil

Xanthoanthrafil

12.Data compiled from published sources.

■1S (USP39)


C173956_160319-M8206-NBDS2015, Rev. 0 20160527

usp–nf | usp-nf - revision bulletin....bulk ingredients • (rb 1-aug-2016) and dosage forms tated...

Documents