Study Of Novel Polar-modified Silica Stationary Phases And Their Application In Pharmaceutical Analysis

Reversed-phase liquid chromatography is the most popular and efficient separationtechnology due to its excellent reproducibility, highly separation efficiency and MScompatibility. Now a very large number of reversed-phase liquid chromatographystationary phases are commercially available on the market and new kinds are beingdeveloped regularly. However, current silica-based LC stationary phases have severalshortcomings such as peak tailing of basic compounds and dewetting in highly aqueousconditions, which limit their use in certain applications. During the past few decades,much work has focused on enhancing the chemical stability of silica-based stationaryphases.Several new stationary phases containing embedded or endcapped polar groupshave been developed, providing users with excellent separation performance for moredifficult chromatographic separations,such as polar, highly basic and ionizable compounds in reversed phase conditions.Aims1. The aim of this research is to design and synthesize three novel C₁₀depeptide polarstationary phases; filter and optimiza the synthesis route.2. The chromatographic performance evaluation of these modified polar stationary phasesis executed by international standard tests.3. To investigate the chromatographic ability to separate and analyze different typepharmaceuticals of these dipeptide polar columns.4. To study the Bioequivalence of Rifapentine.Methods and Results1. Designed and synthesized three target novel C₁₀dipeptide polar stationary phases:We prepared2-undecanamidoacetic acid as intermediates by DCC, DMAPcondensation. Then the acid reacts with N-aminopropyltrimethoxysilane,N-aminopropyldimethylmethoxysilane, N-aminopropyldiisopropylmethoxy-Silane andreceive the corresponding silanes, yield respectively83.3%,85.1%,82.0%. The C₁₀dipeptide silanes are then bonded on silica and react with endcapping reagent ofhexamethyldisilazane. So we obtain three phases: Polymeric C₁₀dipeptide, C₁₀dimethyldipeptide, C₁₀diisopropyl dipeptide stationary phase.2.Evaluated its performance of thethree C₁₀dipeptide stadionary phases, and obtained the expected results.The results of Column performance and peak shape test, Engelhardt test, Tanaka testand Neue test show that the polarity modified dipeptide stationary phases we preparedhave high selectivity, high sensitivity and excellent efficiency. The mixture of pyridine andphenol were separated in the Base deactivation property test, the new C₁₀dipeptidecolumns demonstrated a higher separation factor and a lower tailing factor of pyridine. Itindicated that the effect of endcapped is very good. The results of pH stability test provideconclusive and positive evidence of the stability of the C₁₀dimethyl dipeptide phase inhighly acid and alkali conditions. Successive injections of the same samples1,000times,and results showed less than1%change in retention and peak shape, which indicated thesestationary phases were stable and good reproducibility. The outcome of effects of mobile phase pH on selectivity evinced these new dipeptide stationary phases can be used frompH1.5to pH11.3. Study the application of these C₁₀dipeptide stationary phases in pharmaceuticalanalysis and also got the desired effect.These polar dipeptide stationary phases were applied to separate a variety of mixtures inwider pH and under highly aqueous mobile phase conditions. The results showed that:these could separate: the mixture of category5water-soluble vitamins; the mixture ofcategory8nucleotides; the mixture of α-, δ-,γ-tocopherols; β-blockers; the mixture of4parabens; caffeine metabolites. Results showed that the eight compounds were allobtained excellent separation efficiency.4. The study the Bioequivalence of Rifapentine:20healthy experimenters oral the test formulation（A）600mg and the referenceformulation（R）600mg in the mode of two period and single dose, and then mensurateconcentration of Rifapentine in plasma by HPLC. Control the reference formulation asstandard, the relative bioavailability of the test formulation is91.2%. No significantdifference existed between two Rifapentine in pharmacokinetics parameters, e.g. T_1/2, T_max,C_max, AUC_（0-72h）, and they were bioequivalence in human.