Microfluidic chip-based liquid chromatography coupled to mass spectrometry for determination of small molecules in bioanalytical applications: an update.

Many microfluidic chip-based LC-MS systems have been utilized for high-throughput analysis in various fields of bioanalytical applications such as proteomic, glycomic, pharmaceutical, and clinical research. This review is an update of a previous review article (Electrophoresis 2012, 33, 635-643) to mainly cover the most recent advancements in chip-based LC-MS for determining small molecules in bioanalysis. First, the different types of microfluidic chip devices for chip-based LC-MS analysis will be discussed. Following the discussion of the recent developments in the chip-based instrumentation, the applications of chip-based LC-MS for determining small molecules, such as glycans, pharmaceutical drugs, drugs of abuse, drug metabolites, and biomarkers in various biological sample matrixes will also be included in this review.

Microfluidic chip-based liquid chromatography coupled to mass spectrometry for determination of small molecules in bioanalytical applications.

The development and integration of microfabricated liquid chromatography (LC) microchips have increased dramatically in the last decade due to the needs of enhanced sensitivity and rapid analysis as well as the rising concern on reducing environmental impacts of chemicals used in various types of chemical and biochemical analyses. Recent development of microfluidic chip-based LC mass spectrometry (chip-based LC-MS) has played an important role in proteomic research for high throughput analysis. To date, the use of chip-based LC-MS for determination of small molecules, such as biomarkers, active pharmaceutical ingredients (APIs), and drugs of abuse and their metabolites, in clinical and pharmaceutical applications has not been thoroughly investigated. This mini-review summarizes the utilization of commercial chip-based LC-MS systems for determination of small molecules in bioanalytical applications, including drug metabolites and disease/tumor-associated biomarkers in clinical samples as well as adsorption, distribution, metabolism, and excretion studies of APIs in drug discovery and development. The different types of commercial chip-based interfaces for LC-MS analysis are discussed first and followed by applications of chip-based LC-MS on biological samples as well as the comparison with other LC-MS techniques.

Preparation and evaluation of poly(alkyl methacrylate-co-methacrylic acid-co-ethylene dimethacrylate) monolithic columns for separating polar small molecules by capillary liquid chromatography.

In this study, methacrylic acid (MAA) was incorporated with alkyl methacrylates to increase the hydrophilicity of the synthesized ethylene dimethacrylate-based (EDMA-based) monoliths for separating polar small molecules by capillary LC analysis. Different alkyl methacrylate-MAA ratios were investigated to prepare a series of 30% alkyl methacrylate-MAA-EDMA monoliths in fused-silica capillaries (250-µm i.d.). The porosity, permeability, and column efficiency of the synthesized MAA-incorporated monolithic columns were characterized. A mixture of phenol derivatives is employed to evaluate the applicability of using the prepared monolithic columns for separating small molecules. Fast separation of six phenol derivatives was achieved in 5 min with gradient elution using the selected poly(lauryl methacrylate-co-MAA-co-EDMA) monolithic column. In addition, the effect of acetonitrile content in mobile phase on retention factor and plate height as well as the plate height-flow velocity curves were also investigated to further examine the performance of the selected poly(lauryl methacrylate-co-MAA-co-EDMA) monolithic column. Moreover, the applicability of prepared polymer-based monolithic column for potential food safety applications was also demonstrated by analyzing five aflatoxins and three phenicol antibiotics using the selected poly(lauryl methacrylate-co-MAA-co-EDMA) monolithic column.

Recent developments in microfluidic chip-based separation devices coupled to MS for bioanalysis.

In recent years, the development of microfluidic chip separation devices coupled to MS has dramatically increased for high-throughput bioanalysis. In this review, advances in different types of microfluidic chip separation devices, such as electrophoresis- and LC-based microchips, as well as 2D design of microfluidic chip-based separation devices will be discussed. In addition, the utilization of chip-based separation devices coupled to MS for analyzing peptides/proteins, glycans, drug metabolites and biomarkers for various bioanalytical applications will be evaluated.

Microfluidic chip-based nano-liquid chromatography tandem mass spectrometry for quantification of aflatoxins in peanut products.

Aflatoxins (AFs), a group of mycotoxins, are generally produced by fungi Aspergillus species. The naturally occurring AFs including AFB1, AFB2, AFG1, and AFG2 have been clarified as group 1 human carcinogen by International Agency for Research on Cancer. Developing a sensitive analytical method has become an important issue to accurately quantify trace amount of AFs in foodstuffs. In this study, we employed a microfluidic chip-based nano LC (chip-nanoLC) coupled to triple quadrupole mass spectrometer (QqQ-MS) system for the quantitative determination of AFs in peanuts and related products. Gradient elution and multiple reaction monitoring were utilized for chromatographic separation and MS measurements. Solvent extraction followed by immunoaffinity solid-phase extraction was employed to isolate analytes and reduce matrix effect from sample prior to chip-nanoLC/QqQ-MS analysis. Good recoveries were found to be in the range of 90.8%-100.4%. The linear range was 0.048-16 ng g(-1) for AFB1, AFB2, AFG1, AFG2 and AFM1. Limits of detection were estimated as 0.004-0.008 ng g(-1). Good intra-day/inter-day precision (2.3%-9.5%/2.3%-6.6%) and accuracy (96.1%-105.7%/95.5%-104.9%) were obtained. The applicability of this newly developed chip-nanoLC/QqQ-MS method was demonstrated by determining the AFs in various peanut products purchased from local markets.

Preparation and evaluation of 1,6-hexanediol ethoxylate diacrylate-based alkyl methacrylate monolithic capillary column for separating small molecules.

Due to the high porosity, good thermal stability, and good physical stability at high pressure, polymer monoliths have been successfully utilized as the stationary phases for capillary liquid chromatography (LC) analysis. In this study, we introduced 1,6-hexanediol ethoxylate diacrylate (HEDA) as a cross-linker to prepare alkyl methacrylate monoliths for efficient separation of polar small molecules. HEDA provided additional dipole-dipole interactions between the monolithic stationary phases and polar analytes. For comparison, ethylene dimethacrylate and 1,6-hexanediol dimethacrylate were also utilized as cross-linkers to prepare alkyl methacrylate monoliths. A series of alkyl methacrylate polymeric monoliths were synthesized in fused-silica capillaries using the three different cross-linkers. The porosity, permeability and column efficiency of the synthesized alkyl methacrylate monoliths were characterized. A mixture of phenol derivatives was employed to evaluate the applicability of the prepared monolithic columns for separating small molecules using capillary LC. The HEDA-based alkyl methacrylate monoliths offered the most efficient chromatographic separation for phenol derivatives. Moreover, the capability of applying the novel HEDA-based alkyl methacrylate monolithic columns for potential environmental analysis was demonstrated by separating eight phenylurea herbicides.

Determination of 7-aminoflunitrazepam in urine by dispersive liquid-liquid microextraction with liquid chromatography-electrospray-tandem mass spectrometry.

Dispersive liquid-liquid microextraction (DLLME) and liquid chromatography-electrospray-tandem mass spectrometry (LC-ES-MS/MS) procedure was presented for the extraction and determination of 7-aminoflunitrazepam (7-aminoFM2), a biomarker of the hypnotic flunitrazepam (FM2) in urine sample. The method was based on the formation of tiny droplets of an organic extractant in the sample solution using water-immiscible organic solvent [dichloromethane (DCM), an extractant] dissolved in water-miscible organic dispersive solvent [isopropyl alcohol (IPA)]. First, 7-aminoFM2 from basified urine sample was extracted into the dispersed DCM droplets. The extracting organic phase was separated by centrifuging and the sedimented phase was transferred into a 300 microl vial insert and evaporated to dryness. The residue was reconstituted in 30 microl mobile phase (20:80, acetonitrile:water). An aliquot of 20 microl as injected into LC-ES-MS/MS. Various parameters affecting the extraction efficiency (type and volume of extraction and dispersive solvent, effect of alkali and salt) were evaluated. Under optimum conditions, precision, linearity (correlation coefficient, r(2)=0.988 over the concentration range of 0.05-2.5 ng/ml), detection limit (0.025 ng/ml) and enrichment factor (20) had been obtained. To our knowledge, DLLME was applied to urine sample for the first time.

Online solid-phase extraction liquid chromatography-electrospray-tandem mass spectrometry analysis of buprenorphine and three metabolites in human urine.

An online solid-phase extraction (SPE) liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) method for the determination of buprenorphine (Bup), norbuprenorphine (nBup), buprenorphie-3-beta-d-glucuronide (Bup-3-G) and norbuprenorphie-3-beta-d-glucuronide (nBup-3-G) in human urine was developed and validated. A mixed mode SPE column with both hydrophilic and lipophilic functions was used for online extraction. A C18 column was employed for LC separation and ESI-MS/MS was utilized for detection. Buprenorphine-D(4) (Bup-D4) and norbuprenophine-D3 (nBup-D3) were used as internal standards for quantitative determination. The extraction, clean-up and analysis procedures were controlled by a fully automated six-port switch valve. Identification and quantification were based on the following transitions: m/z 468-->414 for Bup, m/z 414-->364 for nBup, m/z 644-->468 for Bup-3-G and m/z 590-->414 for nBup-3-G, respectively. Good recoveries from 93.6% to 102.2% were measured and satisfactory linear ranges for these analytical compounds were determined. Minimal ion suppression effect (approximately 7% response decrease) was determined. Intra-day and inter-day precision showed coefficients of variance, CV, ranged from 3.3% to 10.1% and 4.4% to 9.8%, respectively. Accuracy ranging from 97.0% to 104.0% was determined. The applicability of this newly developed method was demonstrated by analyzing human urine samples from the patients in Bup treatment program for therapeutic monitoring purpose.

Determination of flunitrazepam and 7-aminoflunitrazepam in human urine by on-line solid phase extraction liquid chromatography-electrospray-tandem mass spectrometry.

A method using an on-line solid phase extraction (SPE) and liquid chromatography with electrospray-tandem mass spectrometry (LC-ES-MS/MS) for the determination of flunitrazepam (FM2) and 7-aminoflunitrazepam (7-aminoFM2) in urine was developed. A mixed mode Oasis HLB SPE cartridge column was utilized for on-line extraction. A reversed phase C(18) LC column was employed for LC separation and MS/MS was used for detection. Sample extraction, clean-up and elution were performed automatically and controlled by a six-port valve. Recoveries ranging from 94.8 to 101.3% were measured. For both 7-aminoFM2 and FM2, dual linear ranges were determined from 20 to 200 and 200-2000ng/ml, respectively. The detection limit for each analyte based on a signal-to-noise ratio of 3 ranged from 1 to 3ng/ml. The intra-day and inter-day precision showed coefficients of variance (CV) ranging from 4.6 to 8.5 and 2.6-9.2%, respectively. The applicability of this newly developed method was examined by analyzing several urine samples.

Determination of amphetamine and methamphetamine in urine by solid phase extraction and ion-pair liquid chromatography-electrospray-tandem mass spectrometry.

A method using a solid phase extraction (SPE) and ion-pair liquid chromatography-electrospray-tandem mass spectrometry (LC-ES-MS/MS) was developed for determination of amphetamine (Amp) and methamphetamine (mAmp) in urine samples. A reversed phase C(18) column was utilized for LC separation and MS/MS was used for detection. Trifluoroacetic acid was added to the mobile phase as an ion-pairing reagent. MS(2) was employed for quantitative determination. In addition, d(8)-amphetamine and d(8)-methamphetamine were used as internal standards. An Oasis HLB SPE cartridge, which has hydrophilic and lipophilic functions, was utilized for sample pre-treatment. Recoveries ranging from 97.3 to 102.1% were measured. Good linear ranges, 5-500 ng/ml, for Amp and mAmp were determined. The detection limit of each analytical compound, based on a signal-to-noise ratio of 3, was approximately 1 ng/ml. The applicability of this newly developed method was examined by analyzing several urine samples from drug users.