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Automation of analytical sample preparation and testing is always attractive due to its high efficiency and continuous operation, but it can also be very challenging due to the operational complexity and method development. Studies focused on method development in automated systems are extremely rare and little has been published on the subject. In this paper, we investigate several unique and advanced aspects of this subject. The example product is selected containing two APIs to be tested by a single method. The product strengths of the two APIs are significantly different, making development of the single method much more challenging. In addition, we apply the additional challenge of using a “green chemistry” approach, avoiding any organic solvent in the dissolving media in order to achieve an environmentally friendly solution. A thorough study on the selection and optimization of several key operational parameters are then discussed. The successful method is achieved using a new strategy called “Automated Online Standard” methodology. This novel approach is compared to the conventional procedure in several scenarios, demonstrating its high effectiveness with significantly improved recovery. The proposed approach can substantially reduce the method development time, generally considered the biggest obstacle to practical deployment of automated systems. The automated system applied in this paper is most suitable for content uniformity testing, but is also applicable for blend uniformity testing, potency assays and degradation product tests.

Relative Response Factors (RRFs) can be used for quantitation of one compound against another and it is widely used for Impurity analysis of pharmaceutical products; however, the application in potency assay is limited. Through an extensive study shown in this paper, it can be concluded that using the “RRF methodology” for potency assay is much more challenging compared to impurity analysis, due to the much tighter criteria required for potency analysis. The effects of instrument settings, which are rarely discussed or recognized in current HPLC analytical method development and quality release testing, are discussed. These factors impact the RRF just as much as other commonly recognized HPLC parameters. The effects of UV detector settings, i.e. Slit Width, Step Width, Band Width, and Data Collection Module, have been explored. This phenomenon has been demonstrated using three compounds to observe the impact of their quantitation due to the significant RRF variations. Finally, principles to reduce RRF variations have been discussed, and practical considerations of RRF application to method development and method transfer are provided.

Automation of analytical sample preparation and testing is always attractive due to its high efficiency and continuous operation, but it can also be very challenging due to the operational complexity and method development. Studies focused on method development in automated systems are extremely rare and little has been published on the subject. In this paper, we investigate several unique and advanced aspects of this subject. The example product is selected containing two APIs to be tested by a single method to be productive. The product strengths of the two APIs are significantly different, making development of a single method much more challenging. In addition, we use a “green chemistry” approach, avoiding any organic solvent in the dissolving media in order to achieve an environmentally friendly solution. The selection and optimization of several key operational parameters are then discussed. The successful method is achieved using a new strategy called “Automated Online Standard (AO-STD)” methodology. This novel approach is compared to the manual operation procedure. The proposed approach can substantially reduce the method transfer time, generally considered the biggest obstacle to practical deployment of automated systems. The automated system applied in this paper is most suitable for content uniformity testing, but is also applicable for potency assays, blend uniformity testing, and degradation product tests.

Benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides that usually contains C-10, C-12, C-14, and C-16 homologues), commonly known as BKC, is used as a bacteriostatic agent in many household, food, and drug products. In this paper, we report a simple, rapid, robust, and stability-indicating reversed-phase HPLC method using a short butyl (C4) column for the simultaneous determination of each individual homologue content, as well as the total concentration of individual homologues in commercial bulk raw material batches of BKC samples. The chromatographic separation was performed on a 5 cm ACE C4 column with mobile phase consisting of water, acetonitrile, and potassium chloride. Even though using a short column can potentially cause some challenges to resolving certain critical pairs of peaks, we have successfully separated all of the analyte peaks (including those from stressed, degraded products) on a short column using an optimal mobile phase.

In this paper, we describe a strategy that can be used to efficiently develop a high-performance liquid chromatography (HPLC) separation of challenging pharmaceutical molecules. This strategy involves use of advanced chromatographic technologies, such as a computer-assisted chromatographic method development tool (ChromSword) and an automated column switching system (LC Spiderling). This process significantly enhances the probability of achieving adequate separations and can be a large time saver for bench analytical scientists. In our study, the ChromSword was used for mobile phase screening and separation optimization, and the LC Spiderling was used to identify the most appropriate HPLC columns. For proof of concept, the analytes employed in this study are the structural epimers betamethylepoxide and alphamethylepoxide (also known as 16-beta methyl epoxide and 16-alpha methyl epoxide). Both of these compounds are used in the synthesis of various active pharmaceutical ingredients that are part of the steroid pharmaceutical products. While these molecules are relatively large in size and contain various polar functional groups and non-polar cyclic carbon chains, their structures differ only in the orientation of one methyl group. To our knowledge, there is no reported HPLC separation of these two molecules. A simple gradient method was quickly developed on a 5 cm YMC Hydrosphere C 18 column that separated betamethylepoxide and alphamethylepoxide in 10 min with a resolution factor of 3.0. This high resolution provided a true baseline separation even when the concentration ratio between these two epimers was 10,000:1. Although outside of the scope of this paper, stability-indicating assay and impurity profile methods for betamethylepoxide and for alphamethylepoxide have also been developed by our group based on a similar method development strategy.

Betamethylepoxide (16beta-methyl-Delta(1,4)-pregnadiene-9beta-11beta-oxide-17alpha,21-diol-3,20-dione) is a key intermediate for the synthesis of various active pharmaceutical ingredients (APIs) of steroid compounds. A stability-indicating reversed-phase HPLC method for assay of betamethylepoxide and estimation of its related compounds has been developed and validated. This method can accurately quantitate betamethylepoxide in the presence of numerous structurally related compounds (including the alpha-epimer, known as alphamethylepoxide). This method can also adequately separate most of the impurities from each other and estimate their quantities in betamethylepoxide samples. The stability-indicating capability of this method has been demonstrated by adequate separation of the degradation products from betamethylepoxide in stress degraded and aged stability samples. The HPLC column used in the method was a 5 cm YMC Hydrosphere C(18) column (4.6 mm I.D.) and the mobile phase consisted of (A) water and (B) acetonitrile:methanol (8:25, v/v).

Use of a novel rotating polycation-sensitive polymer membrane electrode yields sensors that can serve as simple potentiometric titration endpoint detectors for the determination of three FDA approved low-molecular-weight heparin (LMWH) anticoagulant drugs (Fragmin, Normiflo, and Lovenox). The rotating electrode configuration dramatically improves the reproducibility and increases the sensitivity for LMWH determinations by protamine titration. At a rotation speed of 3000 rpm, electrodes with optimized thin (50 microm) polymer membranes doped with dinonylnaphthalene sulfonate (DNNS) respond to low levels of protamine (2 microg mL(-1)) with good precision (+/-1 mV, N=10), when protamine is infused continuously into a Tris-buffer solution, pH 7.4. When infusing protamine (at 5 microg min(-1)) continuously into solutions containing Fragmin, a clear endpoint is obtained, with the amount of protamine required to reach this endpoint proportional to the level of Fragmin present. A detection limit of less than 0.02 U mL(-1) Fragmin can be obtained via this new method, approximately one order of magnitude lower than that previously reported based on a non-rotating polycation electrode. Similar low detection limits can be achieved for potentiometric titrations of Normiflo and Lovenox. Such titrations can also be carried out in undiluted plasma samples containing the various LMWH species. In this case, detection of the LMWHs at clinically relevant concentrations (0.2 U mL(-1)) can be readily achieved.

Deposition of hyperbranched poly(acrylic acid) (PAA) films on porous supports is an attractive method for synthesizing derivatizable, ultrathin composite membrane skins. We previously synthesized these films by sputtering a thin gold layer onto a porous alumina support and then grafting several layers of PAA to a self-assembled monolayer of mercaptoundecanoic acid on the gold. This paper demonstrates grafting of PAA onto PAA/poly(allylamine hydrochloride) (PAH) films that were prepared by alternating polyelectrolyte deposition. This procedure overcomes the inconvenience associated with sputtering of gold and allows synthesis of defect-free membranes using only one grafted PAA layer. Membrane skins consisting of 2.5 bilayers of PAA/PAH plus 1 layer of grafted PAA (total thickness of ∼14 nm) effectively cover the underlying pores of porous alumina supports without filling them, as shown by field-emission scanning electron microscopy (FESEM) images. When derivatized with H2NCH2(CF2)6CF3, these composite memb...

Grafting of poly(acrylic acid) (PAA) or its N,N-dimethylethylenediamine derivative (PAA/DMEDA) onto gold produces electrodes that respond to protamine or heparin. Binding of these polyions to PAA or PAA/DMEDA limits the access of Ru(NH3)63+ to electrodes, decreasing currents in cyclic voltammograms. In 0.1 M NaCl, the electrodes respond linearly to protamine or heparin concentrations of 0.4–3 μg/mL and 0.4–7 μg/mL, respectively. Current decreases are ca. 10-fold greater than those at similar electrodes coated with thioctic acid. Washing with 0.1 M NaCl at pH 1.2 (protamine) or pH 10 (heparin) regenerates electrodes. The heparin sensor still functions in 5% horse serum.

The ion-channel sensor technique was used to determine heparin concentrations in artificial and horse serum with cyclic voltammetry. The sensor is based on self-assembled monolayers (SAMs) of thioctic acid on which protamine is attached as a receptor to control the rate of [Mo(CN)8]4− oxidation or [Fe(CN)6]3− reduction in the presence of heparin. The analyte, heparin, with its negative charges, neutralizes the positive charges on the protamine receptor and at high heparin concentrations provides the electrode surface with an excess of negative charge, thereby repulsing the marker ions from the electrode surface. This decreases the redox currents and makes them a function of the analyte concentration. In artificial serum, a linear concentration range of 0.05–1.5 μg/ml was obtained for the heparin response at a scan rate of 10.24 V/s when [Mo(CN)8]4− was used as marker. Repeated measurements of heparin in artificial and horse serum gave average heparin concentrations of 1.30 and 1.56 μg/ml, respectively, compared to 1.25 μg/ml heparin that was introduced into the serum. Measurements of heparin in horse serum using a fresh electrode for each sample, however, gave an average heparin concentration of 1.21 μg/ml with a standard deviation of 0.026 μg/ml.

The detection of protamine, a polycation, by use of electrodes modified with self-assembled monolayers of thioctic acid is reported. These sensors can detect protamine concentrations as low as 0.5 μg/mL when [Ru(NH3)6]3+ is used as marker and 2.0 μg/mL when [Fe(CN)6]3- is used as marker. Binding of protamine to the thioctic acid monolayers controls the reduction rate of [Ru(NH3)6]3+ and [Fe(CN)6]3- at the electrode surface due to electrostatic attraction or repulsion between these markers and the monolayer, allowing the indirect detection of protamine. In physiological concentrations, the blood electrolytes sodium, potassium, calcium, and magnesium do not interfere. The sensors respond to protamine in diluted horse serum. They are selective for protamine over Polybrene, another polycation that neutralizes the anticoagulant activity of heparin. Protamine once bound to the electrode surface can be removed by washing with 0.1 M KCl of pH 5.1. An increase in the scan rate of cyclic voltammetry decreased the d...

Ion-channel-mimetic sensing based on monolayers of a hydrogen bond-forming bis(thiourea) receptor was used to detect inorganic anions. Receptor monolayers were formed at the air−water interface and subsequently contacted with a highly oriented pyrolytic graphite electrode. Horizontal touch cyclic voltammetry was performed with subphase solutions containing various electroinactive analyte anions and [Fe(CN)6]4- as electroactive marker. Binding of analyte anions to the receptor monolayer was found to inhibit [Fe(CN)6]4- oxidation. The influences of the analyte anions on the cyclic voltammograms were largest for HPO42- and decreased in the order of HPO42- > F- ≈ SO42- > CH3COO- > Cl-, whereas ion-selective electrodes (ISEs) containing the same receptor respond with a selectivity order of Cl- > SO42- > CH3COO- > H2PO4-/HPO42-. Because the bis(thiourea) receptor does not bind to all potentially hydrogen-bonding sites of most of these anions, it is apparent that several of the larger anions, and in particular p...

Selective binding of electroinactive analytes to electrodes that are chemically modified with receptors can be used to control heterogeneous redox reactions of electroactive species. The latter are in this context often called markers because their use allows the indirect determination of the electroinactive analytes with the inherent possibility for chemical signal amplification. Two different approaches can be distinguished. To structurally mimic natural ion-channel proteins, electrodes are modified with artificial receptors having intramolecular channels that can be blocked by formation of inclusion complexes with the analyte. In more abstract analogy to the working principle of ion-channel proteins, binding of usually charged analytes to receptors without intramolecular channels is used to control redox reactions of the marker species on the basis of electrostatic interactions, and, occasionally, of steric repulsion. The versatility and general characteristics of this type of chemically gated sensors are discussed, and specific examples from recent studies are presented.

The potential and limits of hydrogen-bonding ionophores and their use in chemical sensors are discussed. Several hydrogen-bonding bis-thiourea ionophores have been found to complex inorganic anions, among them most strongly H2PO4-. Using such ionophores, ion-selective electrodes for chloride and sulfate have been developed. Furthermore, hosts that bind nucleotides with up to five hydrogen bonds have been synthesized. They have been applied in nucleotide selective electrodes, optodes and voltammetric sensors mimicking ion channels.

Highly preorganized bis-thiourea receptors based on a xanthene spacer selectively (H2PO4− > CH3COO− > Cl−) bind dihyrogenphosphate via multitopic hydrogen bonding, giving stronger complexes with H2PO4− neutral receptor known so far. The high complexation strengths are rationalized by the hydrogen bond donor strength of the thiourea groups and by host preorganization. The hydrogen acceptor strengths of the guest anions and, for small ions, guest solvation explain the observed selectivity of complexation in dimethyl sulfoxide (DMSO).

Alphamethylepoxide (16alpha-methyl-Delta1,4-pregnadiene-9beta-11beta-oxide-17alpha,21-diol-3,20-dione) is a key intermediate for the synthesis of various active pharmaceutical ingredients of steroid compounds. A stability-indicating reversed-phase high-performance liquid chromatographic (RP-HPLC) method for the assay of alphamethylepoxide and estimation of its related compounds has been developed and validated. It can accurately quantitate alphamethylepoxide in the presence of numerous structurally related compounds (including the beta-epimer, known as betamethylepoxide). This method can also adequately separate most of the impurities from each other and estimate their quantities in alphamethylepoxide samples. The stability-indicating capability of this method has been demonstrated by adequate separation of the degradation products from alphamethylepoxide in stress degraded and aged stability samples. A 15 cmx4.6 mm i.d. ACE C18 HPLC column is the primary column used in this method, and a 15 cmx4.6 mm i.d. A Develosil ODS UG column serves as the alternative column. The mobile phase consisted of 10 mM sodium sulfate, 0.05% (v/v) phosphoric acid, and acetonitrile. This method can accurately assay the content of alphamethylepoxide (in a given lot) with a % relative standard deviation of less than one. It can also estimate individual impurities down to 0.05% level compared with the alphamethylepoxide peak in the sample.

Betamethasone (BM) is an active pharmaceutical ingredient (API) or an intermediate which is used to manufacture various finished pharmaceutical products. Betamethasone is also used as a starting material to manufacture other APIs that are related to this steroid family. It is quite a challenging task to separate dexamethasone (DM) peak (the alpha epimer) and other structurally related compounds from BM. A stability-indicating reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed which can separate and accurately quantitate low levels of DM and other related compounds from BM and also from each other. This method was successfully validated for the purpose of conducting stability studies of betamethsone in quality control (QC) laboratories. The stability-indicating capability of this method was demonstrated by adequate separation of DM and all the degradation product peaks from BM peak and also from each other in aged stability samples of betamethasone. A gradient mobile phase system consisting of (A) water:acetonitrile (90:10, v/v) and (B) acetonitrile:isopropanol (80:20, v/v) was used with an ACE Phenyl column (10 cm x 4.6 mm, 3 microm particles, 100 A pore size) and an ultraviolet (UV) detection at 240 nm.

Potentiometric selectivity coefficients, K A,B pot have been collected for ionophore-based ion-selective electrodes (ISEs) for inorganic anions reported during 1988-1998. In addition to the actual numerical values of K A,B pot together with the methods and conditions for their determination, response slopes, linear concentration (activity) ranges, chemical compositions, and ionophore structures for the ISE membranes are tabulated.