Analysis of crude heparin by (1)H NMR, capillary electrophoresis, and strong-anion-exchange-HPLC for contamination by over sulfated chondroitin sulfate.

We previously published a strong-anion-exchange-high performance liquid chromatography (SAX-HPLC) method for the detection of the contaminant over sulfated chondroitin sulfate (OSCS) in heparin sodium active pharmaceutical ingredient (API). While APIs have been processed to remove impurities, crude heparins contain insoluble material, chondroitin sulfates, heparan sulfate, and proteins that may interfere with the recovery and measurement of OSCS. We examined 500MHz (1)H NMR, capillary electrophoresis (CE), and SAX-HPLC to quantify OSCS in crude heparin. Using our standard API protocol on OSCS spiked crude heparin samples; we observed a weight percent LOD and LOQ for the NMR approach of 0.1% and 0.3%, respectively, while the SAX-HPLC method gave values of 0.03% and 0.09%, respectively. CE data was not amenable to quantitative measurement of OSCS in crude heparin. We developed a modified HPLC sample preparation protocol using crude dissolved at the 100mg/mL level with a 2.5M NaCl solution. This SAX-HPLC approach gave a weight percent LOD of 0.02% and a LOQ of 0.07% and had better performance characteristics than that of the protocol used for APIs.

Use of a hydrolytic procedure and spectrometric methods in the structure elucidation of a thiocarbonyl analogue of sildenafil detected as an adulterant in an over-the-counter herbal aphrodisiac.

A sildenafil-related compound was detected in an herbal dietary supplement marketed as an aphrodisiac. The compound was identified as an analogue of sildenafil in which the carbonyl group in the pyrimidine ring of sildenafil was substituted with a thiocarbonyl group, and the methyl group on the piperazine ring was substituted with a hydroxyethyl group. Based on this structure, the compound was named thiohydroxyhomosildenafil. The structure of the compound was established using HPLCIMS, UV spectrometry, electrospray ionization-MS/MS, NMR spectrometry, and a hydrolytic process. One key product of hydrolysis was 1-(2-hydroxyethyl)-piperazine; the identification of this product defined the amine portion of the compound. Another key product of hydrolysis was hydroxyhomosildenafil, generated by hydrolysis of the thiocarbonyl group to a carbonyl group (C = S --> C = O). Hydroxyhomosildenafil was detected as a minor component in the dietary supplement.

Direct intramolecular gas-phase transfer reactions during fragmentation of sildenafil and thiosildenafil analogs in electrospray ionization mass spectrometry.

A study is made of the mass spectral fragmentation pathways of sildenafil, thiosildenafil, and analogous compounds. A prominent gas-phase reaction that occurs during collision-induced dissociation (CID) of thiosildenafil compounds is the transfer of an alkyl group from the piperazine nitrogen atom to the sulfur atom of the thiocarbonyl group. This phenomenon is clearly demonstrated through a comparison of electrospray ionization mass spectral fragmentation patterns of four sildenafil-type compounds and three related thiosildenafil derivatives. Molecular modeling and fragmentation patterns support a direct intramolecular alkyl transfer mechanism rather than an ion-neutral complex mechanism. CID of thiohydroxyhomosildenafil results in a facile hydroxyethyl migration to the sulfur atom followed by a second intramolecular reaction to form a spiro-1,3-oxathiolane ring, which fragments in two directions to generate both carbonyl and thiocarbonyl product ions from this thiocarbonyl compound. While methyl migration to the thiocarbonyl sulfur atom of thiosildenafil is dominant, methyl migration to the carbonyl oxygen atom of sildenafil may occur to a small extent.

Screening of heparin API by near infrared reflectance and Raman spectroscopy.

Near infrared (NIR) reflectance and laser Raman spectra for a set of 69 heparin powder samples obtained from several foreign and domestic suppliers were measured. Both the NIR and Raman spectra of individual heparin API powder samples were correlated with sample compositions determined from response corrected relative peak areas of the capillary electropherograms of the samples using a partial least squares (PLS) regression model. Twenty-eight sample spectra were used to develop PLS models for the three major sample components; heparin, oversulfated chondroitin sulfate (OSCS) and glycosaminoglycans (GAGs). The PLS models were then used to successfully predict the compositions of 41 additional heparin samples. The success of these rapid, nondestructive technologies to identify contamination of heparin with OSCS demonstrates the potential of spectroscopy and chemometrics for screening of processed raw materials. These technologies are meant for screening purposes and not meant to replace either of the methods (capillary electrophoresis and NMR) currently required by USP and FDA.

Analysis of heparin sodium by SAX/HPLC for contaminants and impurities.

A chromatographic method was developed for the detection and quantification of the contaminant oversulfated chondroitin sulfate (OSCS) and the impurity dermatan sulfate in heparin active pharmaceutical ingredient (API). The HPLC analysis of heparin is carried out using a polymer-based strong anion exchange (SAX) column with gradient elution from 0.125 M sodium chloride to 2.5M sodium chloride buffered mobile phase. The limit of detection (LOD) and limit of quantitation (LOQ) for the contaminant OSCS in heparin were determined to be 0.03% and 0.1%, respectively. The LOD and LOQ for dermatan sulfate, an impurity in heparin sulfate, were determined to be 0.1% and 0.8%, respectively. This manuscript is not a policy document and is not intended to replace either of the methods (capillary electrophoresis and NMR) currently required by the FDA.

Detection of undeclared erectile dysfunction drugs and analogues in dietary supplements by ion mobility spectrometry.

An ion mobility spectrometry (IMS) method was developed to screen for the presence of undeclared synthetic erectile dysfunction (ED) drugs or drug analogues in herbal dietary supplements claiming to enhance male sexual performance. Ion mobility spectra of authenticated reference materials including three FDA approved drugs (sildenafil citrate, tadalafil, vardenafil hydrochloride trihydrate) and five previously identified synthetic analogues (methisosildenafil, homosildenafil, piperidenafil, thiosildenafil, thiomethisosildenafil) were measured to determine their reduced ion mobilities (K(0)). All eight compounds exhibited reduced mobilities between 0.8257 and 1.2876 cm(2)/(Vs). Twenty-six herbal products were then screened for the presence of these compounds, and 15 of the 26 products tested positive for the presence of ED drug or drug analogue adulterants based on their reduced ion mobilities. IMS results were compared against the results obtained from an independent LC/MS reference method for the identical samples. Herbal dietary supplements containing adulterants were classified with 100% accuracy and most of the adulterants were correctly identified by a comparison of the K(0) of the adulterant to the K(0) of the authenticated reference material. The results demonstrate that IMS is a viable method for screening herbal dietary supplements for the presence of ED drug or drug analogue adulterants.

Structure elucidation of thioketone analogues of sildenafil detected as adulterants in herbal aphrodisiacs.

Two analogues of sildenafil were detected in herbal dietary supplements marketed as aphrodisiacs. Both compounds were identified as thioketone analogues of sildenafil in which the carbonyl group in the pyrimidine ring of sildenafil was substituted with a thiocarbonyl group. The first compound was identified as thiosildenafil, a compound that has recently been reported as an adulterant in health supplements. The structure of the second compound was established using LC-MS, UV spectroscopy, ESI-MS(n), NMR and a hydrolytic process. A detailed study of the hydrolysis products of sildenafil, thiosildenafil, and the second unknown compound proved that the second compound, named thiomethisosildenafil, had a structure analogous to sildenafil in which the N-methylpiperazine moiety had been replaced with 2,6-dimethylpiperazine and the oxygen atom of the carbonyl group in the heterocyclic ring had been replaced with a sulfur atom. Under the hydrolytic reaction conditions employed in this study, thioketones hydrolyze to ketones (e.g., thiosildenafil-->sildenafil), making this a valuable technique for the structure elucidation of thiosildenafil analogues. Ten herbal dietary supplements, each as a capsule dosage form, were found to contain 8-151 mg of thiomethisosildenafil per capsule, and one herbal dietary supplement was found to contain 35 mg of thiosildenafil per capsule.

Inhibition of Taq polymerase as a method for screening heparin for oversulfated contaminants.

Heparin and low molecular heparins are extensively used in the treatment of a wide range of diseases in addition to their classic anticoagulant activity and can be found coating medical devices such as catheters, stents and filters. Early in 2008, a sharp increase in heparin-associated severe adverse events, including over 80 deaths, was linked to the presence of a contaminant identified as hypersulfated chondroitin sulfate (OS-CS). OS-CS is one of several oversulfated glycosaminoglycans (GAGs) of different origins that can potentially cause similar clinical problems underscoring the need to develop robust screening methods for contaminants in existing and future lots of heparin. This study demonstrates that oversulfated GAGs block the activity of Taq polymerase used for real time PCR. Based on this finding we developed a simple, rapid, sensitive and high throughput screening method to detect and quantify oversulfated chondroitin sulfate (OS-CS) and other potential oversulfated contaminants in commercial lots of heparin. This method requires less than 100 miliUnits (mU) of heparin as starting material, therefore avoiding the need to lyophilize and concentrate samples, and has a limit of detection of <1 ng for all oversulfated GAGs tested.

Stability of the nitrogen mustard mechlorethamine in novel formulations for dermatological use.

Long term stability measurements were made for the nitrogen mustard mechlorethamine HCl at a concentration of 0.02% in six topical formulations: Aquaphor ointment, Transcutol, Labrasol, 10% Transcutol in Aquaphor, 10% Transcutol in Labrasol, and Aquaphilic ointment. The drug decomposed gradually in Aquaphor ointment at room temperature, dropping to 95% in 4 weeks, 85% in 12 weeks, and 78% in 39 weeks. On the other hand, the drug decomposed rapidly in Aquaphilic ointment, giving an assay of less than 20% of its initial concentration after 24h at room temperature. Generally, mechlorethamine HCl was more stable in Aquaphor ointment than in formulations containing Transcutol or Labrasol. However, the addition of the free radical inhibitor, BHT, significantly enhanced the stability of mechlorethamine in Transcutol and Labrasol formulations. Four BHT-stabilized Transcutol and Labrasol formulations gave assays in ranges of 92-99% at the end of 4 weeks, 77-98% at the end of 12 weeks, and 38-93% at the end of 41 weeks.

Modifications and insights into a method for the analysis of the nitrogen mustard mechlorethamine by high-performance liquid chromatography.

Previously, a method was presented for the analysis of mechlorethamine by derivatization of this unstable nitrogen mustard to bis(2-phenylthioethyl)methylamine (PTEMA), a stable compound suitable for analysis by HPLC with UV detection [J.C. Reepmeyer, J. Chromatogr. A, 1085 (2005) 262]. Mechlorethamine HCl served as a reference standard and it was derivatized in situ simultaneously with samples of mechlorethamine HCl in ointment preparations. This paper presents the synthesis of PTEMA on a gram scale, synthesis of its picrate salt, bis(2-phenylthioethyl)methylamine picrate (PTEMAP), and isolation of the picrate as a crystalline solid. PTEMAP may serve as a reference standard replacing the toxic mechlorethamine HCl. Insights into the handling, storage, drying, and hygroscopic properties of mechlorethamine HCl and PTEMAP are discussed. In addition, one step following the derivatization procedure in the original method is recognized as a potential for error, and a procedure relating to the order of addition of reagents is presented to avoid this error. The method has been extended to the analysis of mechlorethamine in aqueous solutions.

Use of liquid chromatography-mass spectrometry and a chemical cleavage reaction for the structure elucidation of a new sildenafil analogue detected as an adulterant in an herbal dietary supplement.

An herbal dietary supplement, marketed as a natural product for the enhancement of sexual function, was analyzed by HPLC with photodiode array and mass spectral detection and found to contain a compound related to the synthetic phosphodiesterase-5 (PDE-5) inhibitors. Based on UV spectra, mass spectra and direct infusion MS(n), the structure of the compound was tentatively identified as a sildenafil analogue in which the sulfonyl group had been replaced with an acetyl group. This new analogue is similar to acetildenafil, a previously reported sildenafil analogue, but differs in that it contains an N-methyl group where acetildenafil contains an N-ethyl group. The structure of the unknown was unequivocally established by chemical cleavage of the phenacylamine group of the molecule to generate N-methylpiperazine; other cleavage products matched those generated from acetildenafil. Since the new compound has one less CH(2) group than acetildenafil, it was named nor-acetildenafil.

Structure elucidation of a novel analogue of sildenafil detected as an adulterant in an herbal dietary supplement.

A new analogue of sildenafil was detected in an herbal dietary supplement, which was sold over the internet and promoted as a product for the enhancement of sexual performance. The structure of the compound was established using LC-MS, UV spectroscopy, MS-MS, and NMR. In addition, the compound was cleaved at its sulfonamide S-N bond yielding a sulfonic acid and an amine, which were independently characterized using LC-MS, GC-MS, and derivatization. The compound, named methisosildenafil, is a novel synthetic analogue of sildenafil in which the N-methylpiperazine moiety has been replaced with 2,6-dimethylpiperazine.

Use of liquid chromatography-mass spectrometry and a hydrolytic technique for the detection and structure elucidation of a novel synthetic vardenafil designer drug added illegally to a "natural" herbal dietary supplement.

An herbal dietary supplement, marketed as a natural product for the enhancement of sexual function, was purchased covertly over the internet. The product was analyzed by LC-MS and found to contain a compound related to synthetic phosphodiesterase 5 (PDE-5) inhibitors. Based on LC with photodiode array and mass spectral detection, along with collision-induced dissociation-mass spectral analysis, the structure of the compound was tentatively identified as a designer drug of vardenafil in which the N-ethylpiperazine ring had been replaced by a piperidine ring. This structure was unambiguously confirmed by acid hydrolysis of both the unknown ("piperidenafil") and vardenafil and comparison of their hydrolysis products by LC-MS and GC-MS. The hydrolytic technique proved to be a useful tool for the structure elucidation of piperidenafil and may be a useful technique for the structure elucidation of other erectile dysfunction designer drugs in the future. The dosage level of piperidenafil in the herbal product was 41 mg per capsule when calculated as the free base.

Separation and detection of the isomeric equine conjugated estrogens, equilin sulfate and delta8,9-dehydroestrone sulfate, by liquid chromatography--electrospray-mass spectrometry using carbon-coated zirconia and porous graphitic carbon stationary phases.

Equilin-3-sulfate and delta8,9-dehydroestrone-3-sulfate are two isomers found in equine conjugated estrogens that differ in structure only by the position of a double bond in the steroid B-ring. These geometric isomers were not resolved on a C18 column during the analysis of conjugated estrogen drug products by LC-MS using acetonitrile-ammonium acetate buffer as the mobile phase. While no separations of these two isomers were observed on C18 or other alkyl-bonded silica based phases using a variety of mobile phase conditions, partial separations were achieved on phenyl bonded silica phases with a resolution of 1.5 on a diphenyl phase, and baseline separations were readily achieved on two carbonaceous phases with resolutions routinely exceeding three on graphitic carbon-coated zirconia (Zr-CARB) and resolutions as high as 19 on porous graphitic carbon (Hypercarb). An examination of a selected few conjugated estrogens in the complex drug substance by LC-MS on Hypercarb is presented.

Analysis of the nitrogen mustard mechlorethamine in topical pharmaceutical preparations by high-performance liquid chromatography.

Mechlorethamine in topical pharmaceutical formulations was derivatized with benzenethiol to form the disubstitution product and analyzed by normal-phase HPLC on silica gel using dibutyl phthalate as an internal standard. The derivatization reaction, purification, and isolation were conveniently performed in a single test tube. Analyses were successfully performed on three types of ointment formulations: anhydrous hydrophobic petrolatum-based ointments, anhydrous hydrophilic ointments, and hydrous hydrophilic ointments. Precision for the analysis of mechlorethamine standard or mechlorethamine in ointments ranged from 0.08 to 0.52% RSD (n = 6). Recoveries from ointments spiked with 0.02% mechlorethamine hydrochloride were 98.4-100.4%. The chromatograms were clean, showing minimal or no interference from ointment excipients or reagents.