• FOCUS: LIFE SCIENCE
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• no.1
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• pp.3.1-3.7
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• 2024

The article discusses the critical role of chromatography in the analysis and purification of proteins in biopharmaceuticals, emphasizing the importance of comprehensive characterization for ensuring their safety and efficacy. It highlights the use of Avantor® ACE® HPLC columns for the separation and purification of proteins, focusing on the analysis of intact proteins using reversed-phase liquid chromatography (RPLC) with fully porous particles. This article also details the application of different mobile phase additives, such as TFA and formic acid, and emphasizes the advantages of using type B ultra-pure silica-based columns for efficiency and peak shape in biomolecule analysis. Additionally, it addresses the challenges of analyzing intact proteins due to slow molecular diffusion and introduces the concept of solid-core (or superficially porous) particles, emphasizing their benefits over traditional porous particles for the analysis of therapeutic proteins. Furthermore, it discusses the development of Avantor® ACE® UltraCore BIO columns, specifically designed for the high-efficiency separation of large biomolecules, such as proteins, and demonstrates their effectiveness in achieving high-resolution separations, even for higher molecular weight proteins like monoclonal antibodies (mAbs). In addition, it underscores the complexity of analyzing and characterizing intact protein biopharmaceuticals, requiring a range of analytical techniques and the use of wide-pore stationary phases, operated at elevated temperatures and with relatively shallow gradients. It highlights the comprehensive range of options offered by Avantor® ACE® wide pore columns, including both fully porous and solid-core particles, bonded with a variety of complementary stationary phase chemistries to optimize selectivity during method development. The use of ultrapure and highly inert base silica is emphasized for enabling the use of lower concentrations of mobile phase modifiers without compromising analyte peak shape, particularly beneficial for LC-MS applications. Then the article concludes by emphasizing the significance of reversed-phase liquid chromatography and its compatibility with mass spectrometry as a valuable tool for the separation and analysis of intact proteins and their closely related variants in biopharmaceuticals.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.2
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• pp.252-261
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• 2006

Four experiments were conducted to investigate the effect of organic or inorganic acid supplementation on the growth performance, nutrient digestibility, intestinal measurements and white blood cell counts of weanling pigs. In growth trial (Exp I), a total of 100 crossbred pigs ({$Landrace{\times}Yorkshire$}${\times}$Duroc), weaned at $23{\pm}2$ days of age and $7.25{\pm}0.10kg$ average initial body weight (BW), were allotted to 5 treatments by body weight and sex in a randomized complete block (RCB) design. Three different organic acids (fumaric [FUA], formic [FOA] or lactic acid [LAA]) and one inorganic acid (hydrochloric acid [SHA]) were supplemented to each treatment diet. Each treatment had 5 replicates with 4 pigs per pen. During 0-3 wk, average daily gain (ADG), average daily feed intake (ADFI) and feed efficiency (G/F ratio) were not significantly different among treatments. However, pigs fed LAA or SHA diet showed improved ADG by 15 or 13% respectively and 12% greater ADFI in both treatments compared to CON diets. Moreover, compared to organic acid treatments, better ADG (p = 0.07) and ADFI (p = 0.09) were observed in SHA diet compared to pigs that were fed the diet containing organic acids (FUA, FOA or LAA). However, during 4-5 wk, no differences in ADG, ADFI and G/F ratio were observed among treatments. Overall, ADG, ADFI and G/F ratio were not affected by acidifier supplementation. Although it showed no significant difference, pigs fed LAA or SHA diets showed numerically higher ADG and ADFI than pigs fed other treatments. In metabolic trial (Exp II), 15 pigs were used to evaluate the effect of acidifier supplementation on nutrient digestibility. The digestibility of dry matter (DM), crude protein (CP), crude fat (CF), crude ash (CA), calcium (Ca) and phosphorus (P) was not improved by acidifier supplementation. Although the amount of fecal-N excretion was not different among treatments, that of urinary-N excretion was reduced in acidsupplemented treatments compared to CON group (p = 0.12). Subsequently, N retention was improved in acid-supplemented groups (p = 0.17). In anatomical trial (Exp III), the pH and $Cl^-$ concentrations of digesta in gastrointestinal (GI) tracts were not affected by acidifier supplementation. No detrimental effect of intestinal and lingual (taste bud) morphology was observed by acidifier supplementation particularly in inorganic acid treatment. In white blood cell assay (Exp IV), 45 pigs were used for measuring white blood cell (WBC) counts. In all pigs after LPS injection, WBC counts had slightly declined at 2 h and kept elevating at 8 h, then returned to baseline by 24 h after injection of lipopolysaccharide (LPS). However, overall WBC counts were not affected by acidifier supplementation. In conclusion, there was no difference between organic and inorganic acidifier supplementation in weanling pigs' diet, however inorganic acidifier might have a beneficial effect on growth performance and N utilization with lower supplementation levels. Furthermore, inorganic acidifier had no negative effect on intestinal measurements and white blood cell counts in weanling pigs. These results suggested that inorganic acidifier might be a good alternative to organic acidifiers in weanling pigs.

• Korean Journal of Environmental Agriculture
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• v.36 no.3
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• pp.201-210
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• 2017

BACKGROUND: The current study was to monitor of 9 veterinary antibiotics (ceftiofur, clopidol, florfenicol, sulfamethazine, sulfamethoxazole, sulfathiazole, tetracycline, tiamulin, and tylosin) in manure using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in positive and negative electrospray ionization mode. METHODS AND RESULTS: Sample preparation was carried out using Mcllvaine buffer and citrate salts to adjust the pH of the sample followed by purification with dispersive solid phase extraction (d-SPE). Separation of analytes during LC-MS/MS analysis was conducted using an Eclipse Plus $C_{18}$ column and the mobile phase was in gradient mode with, 0.1% formic acid and 5 mM ammonium formate in methanol (A) and 0.1% formic acid and 5 mM ammonium formate in distilled water (B). The linearity of the matrix-matched calibrations of all tested antibiotics was good, with $R^2$ determination coefficients ${\geq}0.9920$. The limit of detection (LOD) and quantifications (LOQ) were $0.1-67.0{\mu}g/kg$ and $0.4-200.0{\mu}g/kg$, respectively. Analysis of 13 solid and liquid manure samples taken from the Republic of Korea revealed concentrations less than $0.7{\mu}g/kg$ for tiamulin, $1497.6{\mu}g/kg$ for sulfamethazine. CONCLUSION: To monitor 9 veterinary antibiotics from manure samples in 13 provincial areas throughout the Republic of Korea, an analytical method was developed. The developed method was fully validated and successfully applied for monitoring various veterinary antibiotics in manure samples.

• Journal of Food Hygiene and Safety
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• v.28 no.3
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• pp.247-251
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• 2013

The objective of present study was to develop a simultaneous determination method of 5 medical compounds, including beclomethasone, dexamethasone, prednisolone, ketoprofen, phenylbutazone in foods, using LC-MS/MS. To optimize MS analytical condition of 5 compounds, each parameter was established by MRM mode. The chromatographic separation was achieved on a C18 column successfully, with a mobile phase made up of A (0.1% formic acid) and B (0.1% formic acid in acetonitrile), at a flow rate of 0.3 mL/min for 17 min with a gradient elution. LOD and LOQ of 5 compounds were in the range of 0.40~4.60 ng/mL and 0.81~11.46 ng/mL, respectively. As a result of analyzing the three concentrations of the standard mixture added to blank samples, the results showed that the mean recovery rate of 5 compounds was in the range of 81.52~103.83%, and RSD (%) of Intra- and Inter-day assay were 0.52-10.45. Since relatively fine selectivity, accuracy and reproducibility were shown in this qualified experimental method, it could be utilized efficiently to investigating those 5 compounds to see if it is added to food products illegally.

• Journal of Food Hygiene and Safety
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• v.31 no.5
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• pp.327-334
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• 2016

This study was conducted to establish the standard method for the contents of biotin in milk formulas. To optimize the method, we compared several conditions for liquid extraction, purification and instrumental measurement using spiked samples and certified reference material (NIST SRM 1849a) as test materials. LC-MS/MS method for biotin was established using $C_{18}$ column and binary gradient 0.1% formic acid/acetonitrile, 0.1% formic acid/water mobile phase is applied for biotin. Product-ion traces at m/z 245.1 ${\rightarrow}$ 227.1, 166.1 are used for quantitative analysis of biotin. The linearity was over $R^2=0.999$ in range of $5{\sim}60{\mu}g/L$. For purification, chloroform was used as a solvent for eliminating lipids in milk formula. The linearity was over 0.999 in range of 5~60 ng/mL. The detection limit and quantification limit were 0.10, 0.31 ng/mL. The accuracy and precision of LC-MS/MS method using CRM were 103%, 2.5% respectively. Optimized methods were applied in sample analysis to verify the reliability. All the tested milk formulas were acceptable contents of biotin compared with component specification and standards for nutrition labeling. The standard operating procedures were prepared for biotin to provide experimental information and to strengthen the management of nutrient in milk formula.

• Korean Journal of Food Science and Technology
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• v.44 no.3
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• pp.263-268
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• 2012

Novobiocin is a coumarin-containing antibiotic, and has a longer half-life in various animals than other veterinary medicines. A simple and rapid high-performance liquid chromatography assay for the determination of residual novobiocin levels in chicken, beef and milk has been developed and validated. The separation condition for HPLC/UVD was optimized by a MG II $C_{18}$ (4.6 mm $ID{\times}250$ mm, 5 ${\mu}m$) column with 0.1% formic acid in $H_2O$/0.1% formic acid in Acetonitrile (40/60, v/v) as the mobile phase at a flow rate of 1.0 mL/min and the detection wavelength was set at 340 nm. Residues were extracted from tissue by blending with methanol. After liquid-liquid partitioning, lipid materials were removed with n-hexane and purification as Silica (1 g, 6 mL) cartridge with 10 mL acetone/dichloromethane (10/90, v/v). Limit of quantification and linearity performed by the analytical method were 0.02 mg/kg and 0.999 ($r^2$), and the recovery range was $88.8{\pm}5.6-100.3{\pm}4.4$, $88.8{\pm}7.2-97.0{\pm}3.2$ and $88.1{\pm}4.3-92.8{\pm}3.6%$. It is expected that this analytical method with regards to novobiocin in chicken, beef and milk could be applied as an official method to administer food safety on veterinary medicines.

• Journal of Life Science
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• v.22 no.11
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• pp.1456-1462
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• 2012

Sorafenib is a multikinase inhibitor and an oral anticancer drug approved for the treatment of patients with advanced renal cell carcinoma and those with unresectable hepatocellular carcinoma. The purpose of this study was to develop an efficient method of the determination of sorafenib in human plasma using tandem mass spectrometry coupled with liquid chromatography (LC/MS/MS) and validate the method by the guidelines of the Korean Food and Drug Administration (KFDA). Plasma samples ($100{\mu}l$) were added with chlorantraniliprole as an internal standard and then mixed with the 0.1% formic acid-containing extraction solution composed of isopropyl alcohol and ethyl acetate (1:4, v/v). After centrifugation, the supernatant was concentrated at $45^{\circ}C$ under negative pressure and centrifugal force. The residue was reconstituted with a mobile phase and injected into the HPLC instrument using a reverse phase Waters XTerra$^{TM}$ C18 column (particle size $3.5{\mu}m$). Liquid chromatography was carried out within the run time of 5 min using a mobile phase composed of buffer (0.1% formic acid and 10 mM ammonium formate), methanol, and acetonitrile (1:6:3, v/v/v). The analytes were monitored by tandem mass spectrometry in the multiple reaction monitoring method programmed to detect sorafenib at 'm/z 465.2 ${\rightarrow}$ 252.5' and chlorantraniliprole at 'm/z 484.4 ${\rightarrow}$ 286.2' with positive electrospray ionization mode ($ES^+$). The result showed the proper linearity ($r^2$ > 0.99) over the range of 2,000-5,000 ng/ml with good accuracy (90.7-103.9%) and precision (less than 10%). The newly developed method using LC/MS/MS was validated by the guideline of KFDA and identified as more sensitive compared to the previous methods.

• Journal of Food Hygiene and Safety
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• v.29 no.2
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• pp.131-140
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• 2014

The objective of this study was to develop a simultaneous method of 8 penicillin antibiotics including amoxicillin, ampicillin, cloxacillin, dicloxacillin, nafcillin, oxacillin, penicillin G and penicillin V in meat using LC-MS/MS. The procedure involves solid phase extraction with HLB cartridge and subsequent analysis by LC-MS/MS. To optimize MS analytical condition of 8 compounds, each parameter was established by multiple reaction monitoring in positive ion mode. The chromatographic separation was achieved on a $C_{18}$ column with a mobile phase of 0.05% formic acid and 0.05% formic acid in acetonitrile at a flow rate of 0.2 mL/min for 20 min with a gradient elution. The developed method was validated for specificity, linearity, accuracy and precision in beef, pork and chicken. The recoveries were 71.0~106%, and relative standard deviations (RSD) were 4.0~11.2%. The limit of detection (LOD) and the limit of quantification (LOQ) were 0.003~0.008 mg/kg and 0.01~0.03 mg/kg, respectively, that are below maximum residue limit (MRL) of the penicillins. This study also performed survey of residual penicillin antibiotics for 193 samples of beef, pork and chicken collected from 9 cities in Korea. Penicillins were not found in all the samples except a sample of pork which contained cloxacillin (concentration of 0.08 mg/kg) below the MRL (0.3 mg/kg).

• 월간산업보건
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• s.8
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• pp.4-30
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• 1988

Examination of working environments was conducted to get more detailed data about harmful working environments and to make a contribution to more effective management. Study was carried out on 722 factories located in Taegu city and eight counties in Kyungpook Province, Korea, for a period of one year, from February 1 to December 30, 1986. The total number and proportion of workers exposed to harmful material was 37,697, 45.2% among 83,368 workers. The results according to exposed material were as follows: 1. In the case of noise, proportion of exceeding the 8-hour TLV was 59%, Included were nail-cutting in assembly metal manufacturing industry and weaving process in textile. 2. Dust in mill process of coal manufacturing industries exceeded the TLV of second class of dust at all parts and exceeded the TLV at 6% as a whole.: 3. The fields of industry lower than 70 lux of illumination were storage equipment of food, auto-winder of textile, painting of wood wares and coal mixing, and 44% of all cases was lower than standard. 4. As a result of temperature index investigation(WBGT), about 12% of all sujects exceeded limit value. Included parts were rolling machine and reducing room. 5. In the case of organic solvents, TLV was exceeded at about 8%, The parts exceeded TLV according to materials belonged to this category were as follows. 1) Toluene: adhesive work in assembly metal manufacturing 2) Xylene: printing and paint mixing in chemical manufacturing 3) Methyl ethytl ketone: paint mixing in all parts examined and coating machine partially in chemical manufacturing 4) Methyl isobutyl ketone: printing in chemical manufacturing 5) Acetone: vapor polishing in assembly metal manufacturing 6. Among specified chemical materials, the concentration of HC1 in the air in metal assembly manufacturing factory exceeded TLV. in one of three assembly metal manufacturing examined. Others, such as benzene, acetic acid, formic acid, sodium hydroxide, formalin, ammonia, copper, chromate etc. were lower than TLV in its indoor atmospheric concentration. As a whole, the proportion of exceeding TLV was about 0.8% 7. The concentrations of inorganic lead were lower than TLV in all parts examined. The results of this investigation show the fact that current management of working environments is not satisfactory, and so more active management is needed.

• Asian Journal of Atmospheric Environment
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• v.11 no.4
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• pp.300-312
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• 2017

To improve the understanding of secondary organic aerosol (SOA) formation from the photo-oxidation of anthropogenic and biogenic precursors at the regional background station on Baengnyeong Island, Korea, gas phase and aerosol chemistries were investigated using the Proton Transfer Reaction Time of Flight Mass Spectrometer (PTR-ToF-MS) and the Aerodyne High Resolution Time of Flight Aerosol Mass Spectrometer (HR-ToF-AMS), respectively. HR-ToF-AMS measured fine particles ($PM_1$; diameter of particle matter less than $1{\mu}m$) at a 6-minute time resolution from February to November 2012, while PTR-ToF-MS was deployed during an intensive period from September 21 to 29, 2012. The one-minute time-resolution and high mass resolution (up to $4000m{\Delta}m^{-1}$) data from the PTR-ToF-MS provided the basis for calculations of the concentrations of anthropogenic and biogenic volatile organic compounds (BVOCs) including oxygenated VOCs (OVOCs). The dominant BVOCs from the site are isoprene (0.23 ppb), dimethyl sulphide (DMS, 0.20 ppb), and monoterpenes (0.38 ppb). Toluene (0.45 ppb) and benzene (0.32 ppb) accounted for the majority of anthropogenic VOCs (AVOCs). OVOCs including acetone (3.98 ppb), acetaldehyde (2.67 ppb), acetic acid (1.68 ppb), and formic acid (2.24 ppb) were measured. The OVOCs comprise approximately 75% of total measured VOCs, suggesting the occurrence of strong oxidation processes and/or long-range transported at the site. A strong photochemical aging and oxidation of the atmospheric pollutants were also observed in aerosol measured by HR-ToF-AMS, whereby a high $f_{44}:f_{43}$ value is shown for organic aerosols (OAs); however, relatively low $f_{44}:f_{43}$ values were observed when high concentrations of BVOCs and AVOCs were available, providing evidence of the formation of SOA from VOC precursors at the site. Overall, the results of this study revealed several different SOA formation mechanisms, and new particle formation and particle growth events were identified using the powerful tools scanning mobility particle sizer (SMPS), PTR-ToF-MS, and HR-ToF-AMS.