John C. Reepmeyer

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.

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.

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.

Analysis of crude heparin by 1H 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.

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, Dose Uniformity, and Palatability of Three Counterterrorism Drugs—Human Subject and Electronic Tongue Studies

PurposeThese studies evaluated the ability of common household food and drink products to mask the bitter taste of three selected anti-terrorism drugs.MethodsThree anti-terrorism drugs (doxycycline, ciprofloxacin hydrochloride, and potassium iodide) were mixed with a variety of common household food and drinks, and healthy adult volunteers evaluated the resulting taste and aftertaste. In parallel, the ASTREE Electronic Tongue was used to evaluate taste combinations. Stability of the mixtures over time was monitored, as was the dosage uniformity across preparations.ResultsFoods and drinks were identified that satisfactorily masked the bitter flavor of each drug. Dose uniformity and stability were also acceptable over the range studied, although some combinations were significantly less stable than others. The electronic tongue was able to differentiate between tastes, but ranked masking agents in a different order than human volunteers.ConclusionsDoxycycline, potassium iodide, and ciprofloxacin, which are stockpiled in solid tablet form, can conveniently be prepared into more palatable formulations, using common household foods and drinks. The electronic tongue can be used to perform an initial screening for palatability.

Separation of R‐ and S‐Thalidomide by Reversed‐Phase HPLC with β‐cyclodextrin in the mobile phase

R- and S-Thalidomide were resolved by reversed-phase HPLC on a C-18 column with β-cyclodextrin in the mobile phase. As the concentration of β-cyclodextrin was increased stepwise from 0 to 20 mM, enantiomeric resolution increased and retention times decreased significantly. The influence of different organic modifiers in the mobile phase were evaluated, and ethanol, among others, proved to be effective. Equilibrium constants for the formation of β-cyclodextrin inclusion complexes of R- and S-thalidomide in EtOH-buffer (5:95) were calculated to be 64 and 76 M−1, respectively.

Detection of clobetasol propionate as an undeclared steroid in zinc pyrithione formulations by high-performance liquid chromatography with rapid-scanning ultraviolet spectroscopy and mass spectrometry

Abstract Clobetasol propionate, an anti-inflammatory glucocorticosteroid, was detected in an over-the-counter topical drug product with no indication on the label of this compound as an ingredient. The product was formulated as a topical spray, a cream, or a shampoo and labeled to contain zinc pyrithione as the active ingredient. The finding of clobetasol propionate in the pharmaceutical products was shown by comparison to an authenticated standard of clobetasol propionate by retention time on normal-phase and reversed-phase HPLC, UV spectroscopy, LC–MS and LC–MS–MS. A simple method was developed and validated for the assay of clobetasol propionate by isocratic reversed-phase HPLC. Several lots contained clobetasol propionate at therapeutic levels of 0.02–0.06%. Zinc pyrithione formulations from two other manufacturers were free of clobetasol propionate.

Enantioseparation of 3-phenylacetylamino-2,6-piperidinedione and related chiral compounds

This paper reports HPLC methodology for the first successful enantiomeric separations of 3-phenylacetylamino-2,6-piperidinedione (PAP), a naturally occurring peptide derivative used for inhibiting the growth of cancer tissues. The chiral separation of four related hydrolysates is also described. A commercially available tris-4-methylbenzoate cellulose (Chiralcel OJ) column was used as the chiral stationary phase, operated in the normal-phase, mode. The results demonstrated that hydrolyzed products of PAP, each of which has a carboxylic acid functionality present in its structure, eluted in a reasonable time and are enantiomerically resolved only when a trace amount of organic acid is present in the mobile phase. Different alcohols (ethanol and isopropanol) and acid additives (trifluoroacetic acid, trichloroacetic acid and acetic acid) were evaluated. In general, for the separation of the acidic enantiomers, ethanol is superior to isopropanol and stronger acids enhance the resolution more effectively. However, chiral separation of PAP could only be accomplished with isopropanol in the mobile phase and no acidic additive was needed.

Effect of Heparin Contaminated with Oversulfated Chondroitin Sulfate on the Collection and Analysis of Plasma~!2009-10-12~!2010-02-04~!2010-03-25~!

Oversulfated chrondroitin sulfate (OSCS) was recently identified as a contaminant of heparin and was associ- ated with serious adverse events in patients treated with heparin. Because heparin is a common component of blood col- lection tubes, we tested the effect of OSCS on the laboratory analysis of plasma. Blood from healthy volunteers (N=50) was collected into tubes containing various mixtures of heparin and OSCS. Samples were inspected for microclots and were analyzed for a panel of 28 routine laboratory tests. No microclots were observed in tubes that contained only heparin but were detected in 18%, 88% and 76% of plasma samples containing 5%, 15%, 20% OSCS (%weight relative to hepa- rin), respectively. OSCS at the highest dose (20%) caused a systematic bias for the following 6 tests: Lactate Dehydro- genase: 18% (12% to 24%); Triiodothyronine: -5.7% (-8.1% to -3.3%); Potassium: -2.8% (-4.2% to -1.4%); Total Protein: 2.5% (1.4% to 3.6%); Chloride: -1.4% (-1.8% to -1.0%) and Uric Acid: 1% (0.5% to 1.4%). In summary, OSCS contami- nation of heparin was found to potentially affect the anticoagulation of plasma and the analytical performance of several routine clinical laboratory tests.