title: Class modeling analysis of heparin 1H NMR spectral data using the soft independent modeling of class analogy and unequal class modeling techniques.
abstract: To differentiate heparin samples with varying amounts of dermatan sulfate (DS) impurities and oversulfated chondroitin sulfate (OSCS) contaminants, proton NMR spectral data for heparin sodium active pharmaceutical ingredient samples from different manufacturers were analyzed using multivariate chemometric techniques. A total of 168 samples were divided into three groups: (a) Heparin, [DS] ≤ 1.0% and [OSCS] = 0%; (b) DS, [DS] > 1.0% and [OSCS] = 0%; (c) OSCS, [OSCS] > 0% with any content of DS. The chemometric models were constructed and validated using two well-established methods: soft independent modeling of class analogy (SIMCA) and unequal class modeling (UNEQ). While SIMCA modeling was conducted using the entire set of variables extracted from the NMR spectral data, UNEQ modeling was combined with variable reduction using stepwise linear discriminant analysis to comply with the requirement that the number of samples per class exceed the number of variables in the model by at least 3-fold. Comparison of the results from these two modeling approaches revealed that UNEQ had greater sensitivity (fewer false positives) while SIMCA had greater specificity (fewer false negatives). For Heparin, DS, and OSCS, respectively, the sensitivity was 78% (56/72), 74% (37/50), and 85% (39/46) from SIMCA modeling and 88% (63/72), 90% (45/50), and 91% (42/46) from UNEQ modeling. Importantly, the specificity of both the SIMCA and UNEQ models was 100% (46/46) for Heparin with respect to OSCS; no OSCS-containing sample was misclassified as Heparin. The specificity of the SIMCA model (45/50, or 90%) was superior to that of the UNEQ model (27/50, or 54%) for Heparin with respect to DS samples. However, the overall prediction ability of the UNEQ model (85%) was notably better than that of the SIMCA model (76%) for the Heparin vs DS vs OSCS classes. The models were challenged with blends of heparin spiked with nonsulfated, partially sulfated, or fully oversulfated chondroitin sulfate A, dermatan sulfate, or heparan sulfate at the 1.0, 5.0, and 10.0 wt % levels. The results from the present study indicate that the combination of (1)H NMR spectral data and class modeling techniques (viz., SIMCA and UNEQ) represents a promising strategy for assessing the quality of commercial heparin samples with respect to impurities and contaminants. The methodologies show utility for applications beyond heparin to other complex products.
authors.affiliation: Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine & Dentistry of New Jersey, Piscataway, New Jersey 08854, USA.
authors.forename: Qingda
authors.initials: Q
authors.lastname: Zang
authors.forename: David A
authors.initials: DA
authors.lastname: Keire
authors.forename: Richard D
authors.initials: RD
authors.lastname: Wood
authors.forename: Lucinda F
authors.initials: LF
authors.lastname: Buhse
authors.forename: Christine M V
authors.initials: CM
authors.lastname: Moore
authors.forename: Moheb
authors.initials: M
authors.lastname: Nasr
authors.forename: Ali
authors.initials: A
authors.lastname: Al-Hakim
authors.forename: Michael L
authors.initials: ML
authors.lastname: Trehy
authors.forename: William J
authors.initials: WJ
authors.lastname: Welsh
mesh_terms: D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D004364:Pharmaceutical Preparations / Q000032:analysis; D011522:Protons
chemical_list: D004364:Pharmaceutical Preparations; D011522:Protons