An official website of the United States government
A method using strong anion exchange solid phase extraction (SAX-SPE) followed by liquid chromatography ultraviolet detection (LC-UV) for the analysis of phenylbutazone (PBZ) and its metabolite oxyphenbutazone (OPBZ) in equine plasma for doping control in horse racing has been developed. By using SAX-SPE, commonly regulated non-steroidal anti-inflammatory drugs (NSAIDs) by the United States Equestrian Federation (USEF), i.e. PBZ, OPBZ, diclofenac, flunixin, ketoprofen, meclofenamic acid and naproxen, and an internal standard, i.e. tolfenamic acid, were first selectively extracted. Then, baseline separation of PBZ, OPBZ from other NSAIDs, internal standard, and residual components of equine plasma was achieved using LC-UV. Finally, PBZ and OPBZ in equine plasma were quantified after an internal calibration curve was created.
more »
« less
Quantification of naproxen in equine plasma for doping control in horse racing using strong anion exchange solid phase extraction followed by liquid chromatography with UV detection
A method using strong anion exchange solid phase extraction (SAX-SPE) followed by liquid chromatography ultraviolet detection (LC-UV) for the analysis of flunixin in equine plasma for doping control in horse racing has been developed. By using SAX-SPE, commonly regulated non-steroidal anti-inflammatory drugs (NSAIDs) by the United States Equestrian Federation (USEF), i.e. PBZ, OPBZ, diclofenac, flunixin, ketoprofen, meclofenamic acid and naproxen, and an internal standard, i.e. flurbiprofen, were first selectively extracted. Then, baseline separation of flunixin from other NSAIDs, the internal standard, and residual components of equine plasma was achieved using LC-UV. Finally, flunixin in equine plasma was quantified after an internal calibration curve was created.
more »
« less
- Award ID(s):
- 1827209
- PAR ID:
- 10173902
- Date Published:
- Journal Name:
- 111th Illinois State Academy of Science Annual Meeting
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
After attending this presentation, attendees will gain knowledge in the strategy to achieve high-throughput and simultaneous analysis of cannabinoids and appreciate a validated LC-UV method for analysis of twelve cannabinoids in hemp oil. This presentation will first impact the forensic science community by introducing three fast LC separations of twelve cannabinoids that can be used with either UV or mass spectrometric (MS) detection. It will further impact the forensic science community by introducing a validated LC-UV method for high-throughput and simultaneous analysis of twelve cannabinoids in hemp oil, which can be routinely used by cannabis testing labs. In recent years, the use of products of Cannabis sativa L. for medicinal purposes has been in a rapid growth, although their preparation procedure has not been clearly standardized and their quality has not been well regulated. To analyze the therapeutic components, i.e. cannabinoids, in products of Cannabis sativa L., LC-UV has been frequently used, because LC-UV is commonly available and usually appropriate for routine analysis by the cannabis growers and commercial suppliers. In the literature, a few validated LC-UV methods have been described. However, so far, all validated LC-UV methods only focused in the quantification of eleven or less cannabinoids. Therefore, a method able to simultaneously analyze more cannabinoids in a shorter run time is still in high demand, because more and more cannabinoids have been isolated and many of them have shown medicinal properties. In this study, the LC separation of twelve cannabinoids, including cannabichromene (CBC), cannabidiolic acid (CBDA), cannabidiol (CBD), cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), cannabigerolic acid (CBGA), cannabigerol (CBG), cannabinol (CBN), delta-8 tetrahydrocannabinol (Δ8-THC), delta-9 tetrahydrocannabinolic acid A (Δ9-THCA A), delta-9 tetrahydrocannabinol (Δ9-THC), and tetrahydrocannabivarin (THCV), has been systematically optimized using a Phenomenex Luna Omega 3 µm Polar C18 150 mm × 4.6 mm column with regard to the effects of the type of organic solvent, i.e. methanol and acetonitrile, the content of the organic solvent, and the pH of the mobile phase. The optimization has resulted in three LC conditions at 1.0 mL/minute able to separate the twelve cannabinoids: 1) a mobile phase consisting of water and methanol, both containing 0.1% formic acid (pH 2.69), with a gradient elution at 75% methanol for the first 3 minutes and then linearly increase to 100% methanol at 12.5 minutes; 2) a mobile phase consisting of water and 90% (v/v) acetonitrile in water, both containing 0.1% formic acid and 20 mM ammonium formate (pH 3.69), with an isocratic elution at 75% acetonitrile for 14 minutes; and 3) a mobile phase consisting of water and 90% (v/v) acetonitrile in water, both containing 0.03% formic acid and 20 mM ammonium formate (pH 4.20), with an isocratic elution at 75% acetonitrile for 14 minutes. In order to demonstrate the effectiveness of the achieved LC separations, a LC-UV method is further validated for the high-throughput and simultaneous analysis of twelve cannabinoids. The method used the mobile phase at pH 3.69, which resulted in significant improvement in throughput compared to other validated LC-UV methods published so far. The method used flurbiprofen as the internal standard. The linear calibration range of all the cannabinoids were between 0.1 to 25 ppm with R2≥0.9993. The LOQ (S/N=10) of the cannabinoids was between 17.8 and 74.2 ppb. The validation used a hemp oil containing 3.2 wt% CBD and no other cannabinoids, which was reported by the vendor with a certificate of analysis, as the matrix to prepare control samples: the hemp oil was first extracted using liquid-liquid extraction (LLE) with methanol; cannabinoids were then spiked into the extract at both 0.5 ppm and 5 ppm level. Afterwards, the recovery, precision (%RSD) and accuracy (%Error) of the control samples were assessed and the results met the requirements by the ISO/IEC 17025 and ASTM E2549-14 guidelines.more » « less
-
Abstract Capillary electrophoresis methods for the separation of carbohydrates with four different detection techniques, namely direct UV, indirect UV, capacitively coupled conductivity, and laser-induced fluorescence detection, were tested and their performance was evaluated and compared in terms of linearity, limits of detection and quantitation, repeatability, recovery, analysis time, and sample treatment. The test set of analytes comprised sucrose, glucose, and fructose. The effect of using lactose as an internal standard on the individual methods was investigated, too. The results showed that laser-induced fluorescence detection is a technique of choice for applications requiring the detection of very low amounts of reducing carbohydrates. Contactless conductivity detection is favorable when detection sensitivity is not a crucial parameter but fast and reliable analysis is required. When only a UV detector is available as a standard part of capillary electrophoresis instruments, direct UV detection can be used when analysis time is not a critical parameter. For fast analysis with UV detectors, indirect UV detection is the technique of choice. Finally, to verify the applicability of the tested methods, samples of cola beverage, honey, and orange juice were analyzed and the results obtained by all four methods were compared. Graphical abstractmore » « less
-
ABSTRACT Peptides are widely used in biomaterials due to their ease of synthesis, ability to signal cells, and modify the properties of biomaterials. A key benefit of using peptides is that they are natural substrates for cell‐secreted enzymes, which creates the possibility of utilizing cell‐secreted enzymes for tuning cell–material interactions. However, these enzymes can also induce unwanted degradation of bioactive peptides in biomaterials, or in peptide therapies. Liquid chromatography–mass spectrometry (LC–MS) is a widely used, powerful methodology that can separate complex mixtures of molecules and quantify numerous analytes within a single run. There are several challenges in using LC–MS for the multiplexed quantification of cell‐induced peptide degradation, including the need for nondegradable internal standards and the identification of optimal sample storage conditions. Another problem is that cell culture media and biological samples typically contain both proteins and lipids that can accumulate on chromatography columns and degrade their performance. Removing these constituents can be expensive, time‐consuming, and increases sample variability. However, loading unpurified samples onto the column without removing lipids and proteins will foul the column. Here, we show that directly injecting complex, unpurified samples onto the LC–MS without any purification enables rapid and accurate quantification of peptide concentration and that hundreds of LC–MS runs can be done on a single column without significantly diminishing the ability to quantify the degradation of peptide libraries. To understand how repeated injections degrade column performance, a model library was injected into the LC–MS hundreds of times. It was then determined that column failure is evident when hydrophilic peptides are no longer retained on the column and that failure can be easily identified by using standard peptide mixtures for column benchmarking. In total, this work introduces a simple and effective method for simultaneously quantifying the degradation of dozens of peptides in cell culture. By providing a streamlined and cost‐effective method for the direct quantification of peptide degradation in complex biological samples, this work enables more efficient assessment of peptide stability and functionality, facilitating the development of advanced biomaterials and peptide‐based therapies.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Conference Paper:
- The DOI is not currently available.
