Compact capillary high performance liquid chromatography system for pharmaceutical on-line reaction monitoring.

Due to their size, conventional high performance liquid chromatographs (HPLCs) are difficult to place close to a reaction vessel within a pharmaceutical manufacturing or development site. Typically, long transfer lines are required to move sample from the reactor to the HPLC for analysis and high solvent usage is required. However, herein a compact and modular separation system has been developed to enable co-location of an HPLC with a small-scale reactor for reaction monitoring in the synthesis of active pharmaceutical ingredients. Using a framework based on capillary HPLC, a compact gradient separation system with a fully modular architecture is described. A custom miniature diode-array detector with a linear dynamic range (up to 1500 mAU at 210 nm) was integrated and evaluated for on-line reaction monitoring. In evaluating system suitability, average peak area %RSD of <3%, and an average retention time %RSD of <0.7%, were achieved. To demonstrate practical utility, the compact system was coupled directly to an on-line lab-scale flow through reactor for continuous reaction monitoring in the laboratory fume hood, where a study of the 3rd Bourne reaction was used to compare the performance of the compact system with a commercially available process HPLC instrument (Waters PATROL UPLC). Further, 33 off-line samples from a continuous crystallization reactor were analysed and it was found that the developed compact HPLC system showed equivalent quantitative performance to an Agilent 1290 Infinity II HPLC system.

Small footprint liquid chromatography-mass spectrometry for pharmaceutical reaction monitoring and automated process analysis.

Liquid chromatography (LC) has broad applicability in the pharmaceutical industry, from the early stages of drug discovery to reaction monitoring and process control. However, small footprint, truly portable LC systems have not yet been demonstrated and fully evaluated practically for on-line, in-line or at-line pharmaceutical analysis. Herein, a portable, briefcase-sized capillary LC fitted with a miniature multi-deep UV-LED detector has been developed and interfaced with a portable mass spectrometer for on-site pharmaceutical analysis. With this configuration, the combined small footprint portable LC-UV/MS system was utilized for the determination of small molecule pharmaceuticals and reaction monitoring. The LC-UV/MS system was interfaced directly with a process sample cart and applied to automated pharmaceutical analysis, as well as also being benchmarked against a commercial process UPLC system (Waters PATROL system). The portable system gave low detection limits (∼3 ppb), a wide dynamic range (up to 200 ppm) and was used to confirm the identity of reaction impurities and for studying the kinetics of synthesis. The developed platform showed robust performance for automated process analysis, with less than 5.0% relative standard deviation (RSD) on sample-to-sample reproducibility, and less than 2% carryover between samples. The system has been shown to significantly increase throughput by providing near real-time analysis and to improve understanding of synthetic processes.

Small-Footprint, Field-Deployable LC/MS System for On-Site Analysis of Per- and Polyfluoroalkyl Substances in Soil.

Per- and polyfluoroalkyl substances (PFASs) are emerging environmental pollutants of global concern. For rapid field site evaluation, there are very few sensitive, field-deployable analytical techniques. In this work, a portable lightweight capillary liquid chromatography (capLC) system was coupled with a small footprint portable mass spectrometer and configured for field-based applications. Further, an at-site ultrasound-assisted extraction (pUAE) methodology was developed and applied with a portable capLC/mass spectrometry (MS) system for on-site analysis of PFASs in real soil samples. The influential variables on the integration of capLC with MS and on the resolution and signal intensity of the capLC/MS setup were investigated. The important parameters affecting the efficiency of the pUAE method were also studied and optimized using the response surface methodology based on a central composite design. The mean recovery for 11 PFASs ranged between 70 and 110%, with relative standard deviations ranging from 3 to 12%. In-field method sensitivity for 12 PFASs ranged from 0.6 to 0.1 ng/g, with wide dynamic ranges (1-600 ng/g) and excellent linearities (R2 > 0.991). The in-field portable system was benchmarked against a commercial lab-based LC-tandem MS (MS/MS) system for the analysis of PFASs in real soil samples, with the results showing good agreement. When deployed to a field site, 12 PFASs were detected and identified in real soil samples at concentrations ranging from 8.1 ng/g (for perfluorooctanesulfonic acid) to 2935.0 ng/g (perfluorohexanesulfonic acid).

Ultraviolet absorbance detector based on a high output power 235 nm surface mounted device-type light-emitting diode.

A new miniaturised capillary flow-through deep-UV absorbance detector has been developed using a microscale surface mounted device- type light-emitting diode (LED) (Crystal IS OPTAN 3535-series), emitting at 235 nm and with a half-height band width of 12 nm, and a high-sensitivity Z-shaped flow-cell. Compared with a previously reported TO-39 ball lens LEDs emitting at 235 nm, the new generation LED produced a 20-fold higher optical output and delivered up to 35 times increase in external quantum efficiency (EQE). The Z-cell was based on a reflective rectangular optical path with cross-sectional dimensions of 100 × 100 µm and a physical optical pathlength of 1.2 mm. Inclusion of UV transparent fused-silica ball lenses, between the SMD and the Z-cell, improved light transmission by a factor of 9 and improved the detector signal-to-noise ratio by a factor of 2.2, at the same input current. The detector was housed within an Al-housing fitted with a cooling fan and demonstrated excellent linearity with stray light down to 0.06%, and an effective pathlength of 1.1 mm (92% of nominal pathlength). The resultant detector was fitted successfully into a briefcase-sized portable capillary HPLC system, and practically demonstrated with the detection of a mixture of 13 test compounds at the sub-mg L-1 level in <5 min using gradient elution.

Miniature Multiwavelength Deep UV-LED-Based Absorption Detection System for Capillary LC.

A new miniature deep UV absorbance detector has been developed using low-cost and high-performance LEDs, which can be operated in both scanning (230 to 300 nm) and individual wavelength (240, 255, and 275 nm) detection modes. The detector is mostly composed of off-the-shelf components, such as LEDs, trifurcated fiber optic assembly, a capillary Z-type flow cell, and photodiodes. It has been characterized for use with a standard capillary LC system and was benchmarked against a standard variable wavelength capillary LC detector. The detector shows very low levels of stray light (<0.4%), utilization of up to 99.0% of the effective path length of the flow cell, a wide dynamic range (0.5 to 200 µg/mL for sulfamethazine, carbamazepine, and flavone), and low noise levels (at 300 µAU level). The detector was applied within a miniaturized LC system.

Modular, cost-effective, and portable capillary gradient liquid chromatography system for on-site analysis.

This work demonstrates the development of a compact, modular, cost-effective separation system configured to address a specific separation problem. The principles of the separation are based on gradient capillary liquid chromatography where the system consists of precision stepper motor-driven portable syringe pumps with interchangeable glass syringes (100 µL to 1000 µL). Excellent flow-rate precision of < 1% RSD was achieved with typical flow-rates ranging from 1 µL/min to 100 µL/min, which was ideal for capillary columns. A variable external loop volume and electrically actuated miniature injection valve was used for sample introduction. Detection was based upon a commercial Z-type UV absorbance flow-cell housed within a custom-built cooling enclosure (40 mm x 40 mm) which also contained a UV-LED light-source and a photodiode. System and chromatographic performance was evaluated using linear gradient elution, with day to day repeatability of <1.5% RSD (n = 6) for peak area, and < 0.4% RSD (n = 6) for retention time, for the separation of a 5 component mixture using a 50 mm X 530 µm ID C18 3 µm particle capillary column. The system can run any commercial or in-house packed columns from 50 mm to 100 mm length with IDs ranging from 200 to 700 µm. The developed portable system was operated using custom-built windows-based chromatography software, complete with data acquisition and system control.

Development of polydimethylsiloxane-microdiamond composite materials for application as sorptive devices.

The development and application of non-porous and porous sorptive rods, comprised of polydimethylsiloxane-microdiamond (PDMS-MD) composites, is reported. The PDMS-MD composites were made porous using inorganic salt (NaCl and NaHCO3) particles as dissolvable templates. Materials with pore size of ~40 µm down to ~5 µm were produced. The advantages of incorporating up to ~60%microdiamond (2-4 µm) into PDMS included: (1) significant increase in the density, which saw the rods sink within the aqueous sample without addition of secondary metal or glass materials, (2) significant improvement in mechanical stability (the porous composite rods could be thermally treated multiple times before application, unlike porous PDMS), (3) increased thermal stability up to 450-500 °C with only 6% weight loss of volatile components, and (4) higher thermal conductivity, estimated to be 108% higher than for PDMS. The PDMS-MD investigated as a sorbent for extraction, followed by liquid desorption and GC-FID analysis. Recovery of the sorbent for test solutes, isoamyl acetate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, and phenethyl acetate, was found to range from ~87% to >100, with RSD of 2.10-12.50% in synthetic wine samples. Non-porous composite rods provided similar % recoveries to a commercial sorptive device (PDMS Twister), whereas porous rods showed improved % recovery for most of the test solutes (>10-20%) when applied under similar conditions. The limits of detection (LOD) for the above solutes within the developed method ranged from 0.60 to 27.30 µg L-1). Application of the PDMS-MD-LD-GC-FID method to white wine samples demonstrated how the PDMS-MD composite material can be applied as a robust and an efficient sorptive phase for trace chemical analysis.

Characterization of large surface area polymer monoliths and their utility for rapid, selective solid phase extraction for improved sample clean up.

While polymer monoliths are widely described for solid phase extraction (SPE), appropriate characterization is rarely provided to unravel the links between physical characteristics and observed advantages and disadvantages. Two known approaches to fabricate large surface area polymer monoliths with a bimodal pore structure were investigated. The first incorporated a high percentage of divinyl benzene (PDVB) and the second explored hypercrosslinking of pre-formed monoliths. Adsorption of probe analytes; anisole, benzoic acid, cinnamic acid, ibuprofen and cortisone were investigated using frontal analysis and the SPE performance was compared with particulate adsorbents. Frontal analysis of anisole described maximum adsorption capacities of 164mgg(-1) and 298mgg(-1) for hypercrosslinked and PDVB adsorbents, respectively. The solvated state specific surface area was calculated to be 341 and 518m(2)g(-1) respectively. BET revealed a hypercrosslinked surface area of 817m(2)g(-1), 2.5 times greater than in the solvated state. The PDVB BET surface area was 531m(2)g(-1), similar to the solvated state. Micropores of 1nm provided the enhanced surface area for hypercrosslinked adsorbents. PDVB displayed a pore size distribution of 1-6nm. Frontal analysis demonstrated the micropores present size exclusion for the larger probes. Recovery of anisole was determined by SPE using 0.4 and 1.0mLmin(-1). Recovery for PDVB remained constant at 90%±0.103 regardless of the extraction flow rate suggesting extraction performance is independent of flow rate. A more efficient sample purification of saccharin in urine was yielded by PDVB due to selective permeation of the small pores.

A simplified approach to direct SPE-MS.

Microextraction by packed sorbent (MEPS) has been directly hyphenated with ESI-MS for the rapid screening of opiates and codeine metabolites in urine. This study introduces a novel format of MEPS that incorporates a two-way valve in the barrel of the syringe enabling the direction of liquid flow to be manipulated. Controlled directional flow (CDF) MEPS allows sharp, concentrated sample bands to be delivered directly to the MS in small volumes and effectively eliminates the need to optimize elution. The method optimization assessed the recovery, matrix effects, and the speed of infusion, all critical variables for optimum ESI performance. Matching extraction workflows demonstrated a reduction in carryover from 65% for conventional MEPS to only 1% for CDF MEPS. The recovery (<89% for 50 µL sample), matrix effects (<42%), linearity (r(2) > 0.99), and LODs (<5 ng/mL) were determined to demonstrate method performance. The optimized approach was employed for the screening of codeine metabolites in urine. The ion trace revealed sharp sample bands corresponding to the codeine metabolites. At-line MEPS-ESI-MS allowed both sample preparation and analysis to be completed in only 5 min facilitating high throughput and alleviating the burden of method development.

Negative ion graphitised carbon nano-liquid chromatography/mass spectrometry increases sensitivity for glycoprotein oligosaccharide analysis.

Negative ion nano-liquid chromatography/mass spectrometry (nano-LC/MS) and tandem mass spectrometry (nano-LC/MS(2)), using graphitised carbon as separating medium, were explored for analysing neutral and acidic O-linked and N-linked oligosaccharide alditols. Compared to the sensitivity of capillary LC/MS (flow rate of 6 microL/min) coupled with a conventional electrospray ionisation source, the nano-LC/MS (flow rate of 0.6 microL/min) with a nanoflow ion source was shown to increase the sensitivity ten-fold with a detection limit in the low-femtomole range. The absolute signals for the [M-nH](n-) ions of the oligosaccharides were increased 100-fold, enabling accumulation of high-quality fragmentation data in MS(2) mode, in which detection of low abundant sequence ions is necessary for characterisation of highly sialylated N-linked oligosaccharides. Oligosaccharides with high numbers of sialic acid residues gave dominant fragments arising from the loss of sialic acid, and less abundant fragments from cleavage of other glycosidic bonds. Enzymatic off-line desialylation of oligosaccharides in the low-femtomole range prior to MS(2) analysis was shown to increase the quality of the spectra. Automated glycofragment mass fingerprinting using the GlycosidIQ software confirmed the oligosaccharide sequence for both neutral desialylated as well as sialylated structures. Furthermore, the use of graphitised carbon nano-LC/MS enabled the detection of four sialylated O-linked oligosaccharides on membrane proteins from ovarian tissue (5 microg of total amount of protein).