Yung-Fong Cheng

Dependence of reversed-phase retention of ionizable analytes on pH, concentration of organic solvent and silanol activity

In reversed-phase chromatography, the retention of ionizable analytes is influenced by the ionic properties of the packing caused by surface silanol groups. We have measured the ion-exchange properties of both reversed-phase bonded phases and their underlying base materials. The probe used in this part of the study was bretylium tosylate. The acquired knowledge is then used for a complete and quantitative understanding of the retention behavior of ionizable compounds as a function of the pH of the mobile phase and the solvent composition. We have studied the retention pattern of a broad range of acids, bases, and polyfunctional analytes over the pH range from 2 to 11 and from water to 80% acetonitrile. A few application examples demonstrate the relevant findings.

Ultrafast liquid chromatography/ultraviolet and liquid chromatography/tandem mass spectrometric analysis

Optimal liquid chromatography/mass spectrometric [LC/MS(/MS)] analysis depends on both the LC selectivity and the electrospray efficiency. Here, we outline a simple and comprehensive LC/MS/MS strategy for the rapid analysis of a wide range of pharmaceutical compounds. To achieve ultrafast LC separation with little sacrifice in peak capacity, one needs to start with a column that provides a good peak capacity at short gradient run times; secondly, it is important to use high flow rates to achieve a good gradient peak capacity. Following this strategy, it was possible to baseline-resolve a mixture (containing acidic, neutral, and basic pharmaceutical analytes) in seconds. By coupling the selectivity provided by fast LC separation with the specificity of MS/MS detection, it is possible to separate and identify a wide range of analytes in 1-min gradient analyses. Also, the impact of mobile phase pH on both the chromatographic selectivity and the MS/MS sensitivity is demonstrated.

Straightforward solid-phase extraction method for the determination of verapamil and its metabolite in plasma in a 96-well extraction plate

Straightforward solid-phase extraction (SPE) methods were developed for the determination of verapamil and its metabolite in a plasma matrix. The spiked plasma sample was pretreated with 2% phosphoric acid followed by two different SPE methods using a Waters Oasis HLB 96-well extraction plate. Recoveries greater than 90% were obtained using both a generic and a selective SPE methods. The generic method is a good starting protocol, and it is applicable to a wide range of compounds. This generic method consists of using 5% methanol as the wash solvent, and 100% methanol for the elution. The limitation of the non-specific method is that it does not remove all plasma constituents that interfere with the quantitation of the metabolite, norverapamil. A second, more specific method was developed using the same Oasis HLB sorbent which removes more plasma interferences and provides cleaner extracts for the HPLC-UV analysis. This selective method uses both the methanol concentration and the pH advantageously to preferentially isolate the analytes of interest from a complex sample matrix. Recoveries of greater than 90% with R.S.D.s less than 3.8% were obtained with this selective method.

Simple and rugged SPE method for the determination of tetracycline antibiotics in serum by HPLC using a volatile mobile phase

SummaryA simple and rugged SPE method for the determination of tetracycline (TC), minocycline (MC) and demeclocycline (DCC) in porcine serum by high performance liquid chromatography (HPLC) was developed. The spiked serum sample was pretreated with 2% phosphoric acid followed by a simple and rugged solid-phase extraction procedure using the OasisTM HLB extraction cartridges. High and reproducible recoveries were obtained even though the cartridges were run dry. The extracted sample analytes were injected onto a Waters SymmetryShieldTM RP8 column. The mobile phase was a simple volatile solution containing 0.1% TFA, 2% methanol and 7% acetonitrile in Water. The antibiotics were detected at 350 nm. The calibration curves were linear from 2.0 to 25.0 μg mL−1 of TC and MC with DCC as the internal standard at a concentration of 25.0 μg mL−1. For six replicate analyses, the average recoveries of TC and MC from porcine serum sample fortified at the level of 2.5 μg mL−1 were 96.1% with 1.3% RSD and 101% with 0.54% RSD; at level of 0.5 μg mL−1 the average recoveries were 88% with 1.6% RSD and 97.8% with 1.4% RSD.

Novel high-performance liquid chromatographic and solid-phase extraction methods for quantitating methadone and its metabolite in spiked human urine

A novel solid-phase extraction (SPE) method and HPLC method were developed for the determination of methadone and its metabolite from spiked human urine. For sample cleanup, a spiked urine sample was pretreated with phosphoric acid followed by a well-thought-out SPE method using a 10-mg Oasis HLB 96-well extraction plate. In this SPE method, the concentration of methanol as well as the pH are optimized to preferentially isolate the analytes of interest from the sample matrix. Low elution volumes (200 microl) are achieved; this eliminates evaporation and reconstitution of the sample solution. Recoveries from human urine matrix were greater than 91% with RSD values less than 4.5%. For the HPLC analysis, the separation was obtained using a SymmetryShield RP18 column with a mobile phase of 0.1% TFA-methanol (60:40, v/v). Good peak shapes were obtained without the need of addition of any competing reagent to the mobile phase. Additionally, significant signal-to-noise enrichment was achieved by diluting the final SPE eluates four-fold with water.

Solid-Phase Extraction for the Determination of Tricyclic Antidepressants in Serum Using a Novel Polymeric Extraction Sorbent

A fast and easy solid-phase extraction method was developed for the determination of amitriptyline, doxepin and their metabolites (nortriptyline and nordoxepin) in porcine serum matrix by high performance liquid chromatography. The spiked serum sample was pretreated with 2% phosphoric acid followed by a simple and rugged SPE procedure using OasisTM HLB extraction cartridges. The SPE method requires only one mL of simple solvent throughout the entire SPE process. High and reproducible recoveries were obtained even though the cartridges ran dry. For five replicate analyses, the average recoveries of parent tricyclic antidepressants and their metabolites were all greater than 94%, and the RSDs were all less than 4.0%.

SIMPLIFIED PROCEDURE FOR THE DETERMINATION OF CODEINE AND ITS METABOLITE IN URINE AND PLASMA BY LC/UV AND LC/MS USING MIXED-MODE CATION EXCHANGE FOR SAMPLE PREPARATION

Novel mixed-mode cation exchange (MCX) and HPLC methods were developed to determine codeine and its metabolite from human urine and porcine plasma. For sample cleanup, spiked sample solutions were acidified followed by a well-thought-out MCX method using 3 cc, 60 mg Oasis® MCX cartridges. In this MCX method, there is no need to condition and to equilibrate the sorbent; the sample is loaded directly onto the sorbent. Recoveries from human urine and porcine plasma matrices were greater than 87% with RSDs less than 5.4%. For the HPLC analysis, the separation was obtained using a Symmetry® C18 column with a simple mobile phase of 0.05% TFA/acetonitrile/methanol at 90:5:5. Good peak shapes were obtained without the need of addition of any competing reagent to the mobile phase.

Chapter 3 - Techniques for sample preparation using solid-phase extraction

This chapter describes techniques for sample preparation using solid-phase extraction (SPE). The 1-D reversed-phase sample clean-up method is a simple technique designed to remove major interferences such as plasma proteins or polar compounds. The 2-D reversed-phase solid-phase extraction method provides much cleaner backgrounds, but the development of the method is more complex. A range of useful methods for the sample preparation of samples of biologic origin (plasma and urine) prior to high-performance liquid chromatography (HPLC) or HPLC and mass spectrometry (MS) or MS analysis are discussed in the chapter, with an emphasis on the general principles of each method. Each method can carried out with multiple analytes and can be adapted to related sample preparation problems without difficulties. However, occasionally, the particular properties of a sample matrix, an analytical technique, or even simply the analytes require departures from the details of the approaches described in the chapter. Both offline and online SPE techniques have advantages depending upon the particular application, and additional progress in the future is anticipated with respect to the speed and efficiency of both approaches.