Journal of Pharmaceutical and Biomedical Analysis 37 (2005) 19–25 Determination of thio-based additives for biopharmaceuticals by pulsed electrochemical detection following HPLC Swati J. Modi a , William R. LaCourse a,∗ , Richard E. Shansky b a Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore 21250, MD, USA b Genzyme Corporation, 45 New York Avenue, P.O. Box 9322, Framingham 01701-9322, MA, USA Received 23 January 2004; accepted 3 August 2004 Available online 28 October 2004 Abstract Pulsed electrochemical detection (PED) following liquid chromatographic separation has been applied to the direct (i.e., without deriva- tization) determination of two major sulfur-containing compounds used as pharmaceutical additives, isopropyl-thio--d-galactopyranoside (IPTG) and monothioglycerol (MTG). Limits of detection of IPTG and MTG were found to be 1 ppb (0.2 pmol, 50 L) and 0.2 ppb (0.1 pmol, 50 L), respectively, using optimized potential-time waveforms applied to a Au electrode. In addition to high sensitivity as compared to optical detection, the simultaneous detection of free thiols and disulfides can be used to study the kinetics of these conversions, as is shown for MTG. A practical application of HPLC–PED is demonstrated in determining MTG in a pharmaceutical formulation. The high selectivity of PED for thiocompounds reduces sample preparation and produces simpler chromatograms in a variety of matrices. © 2004 Elsevier B.V. All rights reserved. Keywords: IPTG; Monothioglycerol; Electrochemistry; Pulsed electrochemical detection; Thiol; Sulfur 1. Introduction Pharmaceutical additives are secondary ingredients that function to enable or enhance the delivery of the primary drug or medicine in a variety of dosage forms, and thereby improve its efficacy, control of bioavailability, uniformity, and/or flow characteristics [1,2]. Additives may also be used for their antimicrobial or antioxidant properties, to extend the shelf life of a product, or to increase the stability of active ingredients [1]. Isopropyl--d-thiogalactopyranoside (IPTG) is used to trigger gene expression that is under control of the lac pro- moter for the overexpression of proteins [3]. IPTG is a chem- ical analog of galactose which cannot be cleaved by the en- zyme -galactosidase. Thus, it can serve as an inducer for activity of the Escherichia coli lac operon by binding and in- activating the lac repressor. Normally, IPTG is used to provide ∗ Corresponding author. Tel.: +1 410 455 2105; fax: +1 410 455 2608. E-mail address: lacourse@umbc.edu (W.R. LaCourse). a means for color detection of recombinant plasmids. During experiments or manufacturing processes, multiple additions of IPTG are often necessary for longer induction times as the compound decays under culture conditions. Monothioglyc- erol (MTG) is a sulfur-containing compound used in buffers and for cellular studies [4–8]. Unlike IPTG, it contains a free thiol group and so it readily converts to its disulfide in solution. Although these compounds are not considered “active” ingredients, they are a part of the manufacturing process and often are a part of the final product. Hence, monitoring their presence and concentration is of critical importance. Several high-performance liquid chromatography (HPLC) methods are available for the determination of thioethers, thiols, disulfides, and other sulfur-containing compounds [9–11]. Since many of these compounds of interest have poor chromophores, the most commonly used method of de- tection is based on the formation of fluorescent derivatives using reagents such as o-phthalaldehyde or N-substituted maleimides [12–14]. These procedures are often labor inten- 0731-7085/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2004.08.042