Photobacterium Species의 lux 오페론에서 발견된 Riboflavin 생합성 유전자들의 기능

이찬용,임종호 한국미생물학회 2002 미생물학회지 Vol.38 No.3

발광 박테리아인 Photobacterium species의 lux 오페론에서 발견된 riboflavin 생합성에 관여하는 유전자들(ribI,II,III,IV)의 기능을 조사하였다. 대장균에서 이들 유전자가 포함된 재조합 플라스미드를 발현시켰을 때 상당량의riboflavin이 합성되는 것을 확인하였으며, 또한 이들 유전자들(ribI,II,III,IV)의 기능을 riboflavin에 대하여 종속 영양체인 대장균 돌연변이주(BSV 11,18)를 이용한 유전학적인 방법과 생화학적 방법으로 분석한 결과, 이들은 각각 riboflavin synthase, 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthase, lumazine synthase, GTP cyclohydrolase II활성도를 갖는 단백질을 코드하는 것으로 밝혀졌다. 이는Photobacterium species의 riboflavin 유전자 체계가 riboflavin 생합성에 관여하는 모든 5개의 유전자들이 한 오페론에 존재하는 Bacillus subtilis와 주요 riboflavin 유전자들이 분리되어 있는 대장균과는 다른, 중간적인 형태를 갖는다는 것을 나타낸다. The functions of riboflavin synthesis genes ( ribI,II,III and IV) found immediately downstream of luxG in the lux operon from Photobacterium species were identified using the biochemical and genetical analysis. The ribI-III gene codes for protein corresponding to that coded by the second (riboflavin synthase), third (3,4-dihydroxy 2-butanone 4-phosphate synthase/GTP cyclohydrolase II) and fourth (lumazine synthase) gene, respectively, of Bacillus subtilis rib operon with the respective gene procuct sharing 41-50% amino acid sequence identity. Unexpectedly, the sequence of the ribIV product of Photobacterium phosphoreum does not correspond in sequence to the protein encoded by the fifth rib gene of Bacillus subtilis. Instead the gene (ribIV) codes for a polypeptide similar in sequence to GTP cyclohydrolase II of Escherichia coli and the carboxy terminal domain of the third rib gene from Bacillus subtilis. Complementation of Escherichia coli riboflavin auxotrophs showed that the function of the gene products of ribII and ribIV are DHBP synthase and GTP cyclohydrolase II, respectively. In addition the experiment, showing that increase in thermal stability of riboflavin synthase coded by ribIon coexpression with ribIII, provided indirect evidence that the latter gene codes for lumazine synthase.

Riboflavin Inhibits Growth of Helicobacter pylori by Down-regulation of polA and dnaB Genes

Hye Jin Kwon,Min Ho Lee,Hyun Woo Kim,Ji Yeong Yang,Hyun Jun Woo,Min Park,Cheol Moon,Sa-Hyun Kim,Jong-Bae Kim 대한의생명과학회 2020 Biomedical Science Letters Vol.26 No.4

Infection of Helicobacter pylori on gastric mucosa is associated with various gastric diseases. According to the WHO, H. pylori causes gastric cancer and has been classified as a class I carcinogen. Riboflavin is an essential vitamin which presents in a wide variety of foods. Previous studies have shown that riboflavin/UVA was effective against the growth inhibition of Staphylococcus aureus, S. epidermidis and multidrug-resistant Pseudomonas aeruginosa and had the potential for antimicrobial properties. Thus, we hypothesized that riboflavin has a potential role in the growth inhibition of H. pylori. To demonstrate inhibitory concentration of riboflavin against H. pylori, we performed agar and broth dilution methods. As a result, we found that riboflavin inhibited the growth of H. pylori. The MIC was 1 mM in agar and broth dilution test. Furthermore, to explain the inhibitory mechanism, we investigated whether riboflavin has an influence on the replication-associated molecules of the bacteria using RT-PCR to detect mRNA expression level in H. pylori. Riboflavin treatment of H. pylori led to down-regulation of polA and dnaB mRNA expression levels in a dose dependent manner. After then, we also confirmed whether riboflavin has cytotoxicity to human cells. We used AGS, a gastric cancer cell line, and treated with riboflavin did not show statistically significant decrease of cell viability. Thus, these results indicate that riboflavin can suppress the replication machinery of H. pylori. Taken together, these findings demonstrate that riboflavin inhibits growth of H. pylori by inhibiting replication of the bacteria.

국내 식품 중 구이, 찜, 볶음, 조림에 존재하는 수용성 비타민 B₁, B₂ 그리고 B₃ 함량 조사

조진주,홍성준,부창국,정유리,정창현,신의철,Cho, Jin-Ju,Hong, Seong Jun,Boo, Chang Guk,Jeong, Yuri,Jeong, Chang Hyun,Shin, Eui-Cheol 한국식품위생안전성학회 2019 한국식품위생안전성학회지 Vol.34 No.5

A conventional Korean meal typically includes various roasted, steamed, stir-fried, and braised foods. For this study, we investigated the contents of water soluble vitamins, $B_1$ (thiamin), $B_2$ (riboflavin) and $B_3$ (niacin) in various roasted, steamed, stir-fried, and braised foods. Method validation for analytical data in this study showed a high linearity ($r^2$>0.999), and the limit of detection and quantification were 0.001-0.067 and $0.002-0.203{\mu}g/mL$, respectively. For accuracy and precision, analytical values using standard reference materials were in the certified ranges. Roasted foods contained 0.039-1.057 mg/100 g of thiamin, 0.058-0.686 mg/100 g of riboflavin and 0.021-21.772 mg/100 g of niacin. Steamed foods contained 0.049-1.066 mg/100 g of thiamin, 0.025-0.548 mg/100 g of riboflavin and 0.134-21.509 mg/100 g of niacin. Stir-fried foods contained 0.114-0.388 mg/100 g of thiamin, 0.014-1.258 mg/100 g of riboflavin and 0.015-2.319 mg/100 g of niacin. Braised foods contained 0.112-1.656 mg/100 g of thiamin, 0.024-0.298 mg/100 g of riboflavin and 0.322-2.157 mg/100 g of niacin. The data on water-soluble vitamins in this study can be used for a nutritional database of conventional Korean meals. 본 연구는 국내 식생활의 기본적인 구성식단으로 알려진 구이, 찜, 볶음, 조림에 존재하는 다양한 영양성분 중 수용성 비타민인 $B_1$ (thiamin), $B_2$ (riboflavin), 그리고 $B_3$ (niacin)의 함량을 확인하였다. 실험분석 타당성 검증을 통해서 높은 직선성($r^2$>0.997)을 확인하였고, 검출한계의 경우 $0.001-0.067{\mu}g/mL$ 그리고 정량한계의 경우 $0.002-0.203{\mu}g/mL$를 확인하였다. 또한 실험값에 대한 정밀도와 반복성에 대한 검증을 위해서 standard reference materials를 통해 실험값과 표준값과의 오차가 신뢰도 이내에 존재한다는 결과를 확인하였다. 본 연구에서 제시된 구이식품의 경우 thiamin은 0.039-1.057 mg/100 g의 범위에 존재하였고, riboflavin은 0.058-0.686 mg/100 g의 범위에 존재했으며, niacin의 경우 0.021-21.772 mg/100 g 의 범위로 나타났다. 찜 종류의 경우 thiamin은 0.049-1.066 mg/100 g의 범위에 존재하였고, riboflavin은 0.025-0.548 mg/100 g의 범위에 존재했으며, niacin의 경우 0.134-21.509 mg/100 g의 범위로 나타났다. 볶음의 경우 thiamin은 0.114-0.388 mg/100 g의 범위에 존재하였고, riboflavin은 0.014-1.258 mg/100 g의 범위에 존재했으며, niacin의 경우 0.015-2.319 mg/100 g의 범위로 나타났다. 조림의 경우 thiamin은 0.112-1.656 mg/100 g의 범위에 존재하였고, riboflavin은 0.024-0.298 mg/100 g의 범위에 존재했으며, niacin의 경우 0.322-2.157 mg/100 g의 범위로 나타났다. 본 연구에서 제시된 구이, 찜, 볶음, 그리고 조림 식품에 대한 수용성 비타민 함량에 대한 영양성분 데이터베이스 구축 연구가 국민 식생활의 중요한 기초자료로 이용될 것으로 확신한다.

Regular moderate exercise training can alter the urinary excretion of thiamin and riboflavin

Young-Nam Kim,Ji Young Choi,Youn-Ok Cho 한국영양학회 2015 Nutrition Research and Practice Vol.9 No.1

BACKGROUND/OBJECTIVES: Physical exercise promotes energy producing pathways requiring thiamin and riboflavin as a coenzyme. Therefore, this study investigated the effects of regular exercise training on urinary excretion of thiamin and riboflavin. MATERIALS/METHODS: Fifty rats were randomly assigned to one of two groups: non-exercise training (NT, n = 25) and regular exercise training (ET, n = 25) for 5 weeks. The rats performed moderate exercise on a treadmill (0.5-0.8 km/hour) for 30 min/day, 5 days/week. Twenty-four hour urine samples were collected at the end of the 0 week, 3rd week, and 5th week of training and thiamin and riboflavin were analyzed. RESULTS: No significant differences in thiamin and riboflavin intakes for each week were observed between the NT and ET groups. Urinary thiamin excretion of each group was the highest at the 5<SUP>th</SUP> week compared to the levels at 0 and 3<SUP>rd</SUP> week. Urinary thiamin at the 5<SUP>th</SUP> week was significantly lower in the ET group than in the NT group. Urinary riboflavin excretion was increased by training duration, however, no difference was observed between NT and ET for each week. At 0 and 3<SUP>rd</SUP> week, no significant relationships were observed between dietary intake and urinary excretion of thiamin and riboflavin, however, at the 5<SUP>th</SUP> week, urinary excretion was significantly increased by dietary intake only in the NT group (P < 0.05). Thiamin excretion of both NT and ET groups was significantly increased with riboflavin excretion at the 5<SUP>th</SUP> week (P < 0.01). CONCLUSION: Regular moderate exercise training increased urinary excretion of thiamin. Dietary intakes and urinary excretions of thiamin and riboflavin showed positive correlation in both the exercise training and non-exercise training groups as the exercise training period went by, while the correlations in the exercise training group were weaker than those in the non-exercise training group. Therefore, regular exercise training can alter the urinary excretion of thiamin and riboflavin in rats.

Riboflavin-mediated RDX transformation in the presence of Shewanella putrefaciens CN32 and lepidocrocite

Bae, S.,Lee, Y.,Kwon, M.J.,Lee, W. Elsevier Scientific Pub. Co 2014 Journal of hazardous materials Vol.274 No.-

The potential of riboflavin for the reductive degradation of a cyclic nitramine, hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), was investigated in the presence of lepidocrocite and/or Shewanella putrefaciens CN32. RDX reduction by CN32 alone or CN32 with lepidocrocite was insignificant, while 110μM RDX was completely reduced by CN32 with riboflavin in 78h. The transformation products identified included nitroso metabolites, formaldehyde, and ammonium, indicating the ring cleavage of RDX. UV and visible light analysis revealed that riboflavin was microbially reduced by CN32, and that the reduced riboflavin was linked to the complete degradation of RDX. In the presence of both CN32 and lepidocrocite (γ-FeOOH), 100μM-riboflavin increased the rate and extent of Fe(II) production as well as RDX reduction. An abiotic study also showed that Fe(II)-riboflavin complex, and Fe(II) adsorbed on lepidocrocite, reduced RDX by 48% and 21%, respectively. The findings in this study suggest that riboflavin-mediated RDX degradation pathways in subsurface environments are diverse and complex. However, riboflavin, either from bacteria or exogenous sources, can significantly increase RDX degradation. This will provide a sustainable clean-up option for explosive-contaminated subsurface environments.

Microbial Peoduction of Riboflavin Using Riboflavin Overproducers, Ashbya gossypii, Bacillus subtilis, and Candida famate: An Overview

Lim, Seong-Han,Park, Jong-Soo,Park, Enoch Y. The Korean Society for Biotechnology and Bioengine 2001 Biotechnology and Bioprocess Engineering Vol.6 No.2

In this paper, the microbial production of riboflavin is reviewed and includes descriptions of riboflavin overproducers, and the biosynthesis and details of the key-enzyme genes related to riboflavin. There kinds of riboflavin overproducers are known; Bacillus subtilis and Candida famate utilize glucose as a carbon source, but the fungus Ashbya gossypii requires plant oil as its sole carbon source. The starting material in ribofalvin biosynthesis is guanosine triphospate (GTP), which is converted to riboflavin through six enzymatic reactions. Though Bacillus subtilis, Candida famate, and Ashbya gossypii operate via different pathways until GTP, they follow the same pathway from GTP to riboflavin. From the metabolic viewpoint, with respect to improved riboflavin production, the supplementation of GTP, aprocess-limiting precursor must be considered. The GTP fluxes originate from three sources, serine, threonine and glyoxylate cycles. The development of pathways to strengthen GTP supplementation using biotechnological techniques remains an issue fro future research.

국내 육성 잡곡에 함유된 Thiamine, Riboflavin 및 Niacin 함량

전진수(Jinsoo Jeon),이상훈(Sang Hoon Lee),최용민(Young Min Choi),정명근(Myoung-Gun Choung) 한국식품영양과학회 2021 한국식품영양과학회지 Vol.50 No.12

잡곡은 영양학적 이점으로 인해 많은 연구가 수행되고 있으나, 국내에서 육성된 잡곡류의 수용성 비타민 함량 정보는 부족한 실정이다. 따라서 본 연구는 국내 육성 잡곡류에 함유된 수용성 비타민 thiamine, riboflavin 및 niacin 함량을 HPLC를 이용하여 정량 분석하였으며, 주성분분석을 통해 분류 특성을 검토하였다. Thiamine 및 riboflavin이 가장 많이 함유된 잡곡은 기장이었으며, niacin이 가장 많이 함유된 잡곡은 보리였다. 반면 옥수수는 thiamine 및 niacin 함량이 가장 낮은 잡곡이었다. 본 결과를 주성분분석으로 검토한 결과, 잡곡류의 종류에 따라 서로 다른 그룹으로 구분이 되는 것을 확인하였다. 다만, 옥수수는 품종에 따른 riboflavin 및 niacin 함량 변이가 큰 잡곡이었으며, 기장과 그룹이 overlap 되는 양상임을 확인하였다. 본 연구결과는 향후 국가표준식품성분표 제10개정판 출간에 필요한 데이터베이스로 활용될 것이다. Although many studies have been conducted worldwide on various nutritional benefits of minor cereals, there is insufficient information on the content of water-soluble vitamins in Korean minor cereal (barley, foxtail millet, maize, oat, proso millet, and sorghum) cultivars. This study therefore quantitatively analyzed the content of water-soluble vitamins, like thiamine, riboflavin, and niacin contained in Korean minor cereals using an high-performance liquid chromatography (HPLC), and the classification characteristics were reviewed through principal component analysis (PCA). Foxtail millet used in this study had the highest thiamine and riboflavin content, and barley contained the most niacin among minor cereals. Among the minor cereals reviewed in this study, maize had the lowest thiamine and niacin. Thereafter, as a result of a PCA review, it was possible to confirm the classification of thiamine, riboflavin, and niacin and to separate them into different groups according to the kinds of minor cereals. However, maize was a minor cereal with a large variation in riboflavin and niacin content depending on the cultivar, and its classification overlapped with proso millet. The results of this study provide a database necessary for the publication of the 10th edition of the Korean Food Composition Table.

역상 HPLC-자외선 및 형광검출기를 이용한 국내 수산자원의 비타민 B₁, B₂ 및 B₃ 분석 및 검증

정보미(Bomi Jeong),박수진(Su-Jin Park),박예은(Ye-Eun Park),곽유정(Yu-Jeong Gwak),김정(Jeong Kim),윤나영(Na-Young Yoon),김연계(Yeon-Kye Kim),천지연(Jiyeon Chun) 한국식품영양과학회 2021 한국식품영양과학회지 Vol.50 No.12

본 연구는 국내 수산자원에 대하여 HPLC-PDA와 HPLCFLD 분석법을 이용한 수용성 비타민 B₁, B₂ 및 B₃ 활성을 나타내는 thiamin, FAD, FMN, riboflavin, nicotinic acid 및 nicotinamide 6종 분석에 대한 분석법 검증과 분석품질관리를 수행하고, 검증된 분석법을 적용하여 수산자원 20종에 대한 신뢰도가 확보된 비타민 B₁, B₂ 및 B₃ 데이터베이스를 구축하고자 수행되었다. 수산자원의 수용성 비타민 분석법 검증은 분석수행지표(특이성, 직선성, 민감성, 정확성 및 정밀성)를 이용하여 평가하였다. 비타민 B₁과 B₃의 표준용액과 수산자원 시료를 분석 비교한 결과 PDA spectrum과 HPLC retention time이 모두 일치하여 분석의 특이성을 확인하였다. 비타민 B₁, B₂ 및 B₃ 모든 성분에서 넓은 범위에서의 분석 직선성(R²=0.9989~1.0000)이 확인되었다. 비타민 B₁(thiamin)의 LOQ는 0.0010 ㎎/100 g을 보였으며, 비타민 B₂(FAD, FMN, riboflavin)의 LOQ는 각각 0.00014, 0.00006, 0.00001 ㎎/100 g을 나타내었다. 또한, 비타민 B₃(nicotinic acid와 nicotinamide)의 LOQ는 각각 0.0008과 0.0021 ㎎/100 g을 나타내어 수산자원의 낮은 수준으로 존재하는 수용성 비타민을 정량 가능한 것으로 확인되었다. 표준참고물질 SRM을 이용한 회수율 측정 결과 98.5~105.8%로 높은 수준을 보였으며, 비타민 B₁의 반복성과 재현성은 각각 5.4%와 8.0%, 비타민 B₂는 각각 3.5%와 4.2%, 비타민 B₃는 각각 3.1%와 5.9%를 나타내어 분석법의 정확성과 정밀성의 우수함이 확인되었다. 수산자원 20종의 비타민 B₁, B₂ 및 B₃ 함량은 각각 0.00~0.84, 0.05~0.47 및 0.00~11.96 ㎎/100 g의 범위를 나타냈으며, 수산자원 6군으로부터의 thiamin, nicotinic acid, nicotinamide, FAD, FMN 및 riboflavin 6종 분석 회수율은 79.9~120.1%의 범위를 나타냈는데, 이는 AOAC 가이드라인에서 제시하는 수용범위 수준을 충족하는 것으로 확인되었다. 내부 분석품질 관리도표와 외부 분석 숙련도 시험 결과를 통해 우수한 숙련도와 분석품질이 분석 전 기간 관리하에 진행되었음이 확인되어 분석 데이터의 신뢰성이 우수함을 확인할 수 있었다. 따라서 본 연구에서 분석한 수산자원 20종의 비타민 B₁, B₂ 및 B₃ 분석 데이터는 국내 수산자원 데이터베이스 구축을 위한 자료로 활용될 수 있을 것으로 판단되며, HPLC/PDA를 이용한 비타민 B₁과 B₃ 동시분석법과 HPLC/FLD를 이용한 비타민 B₂ 분석법은 향후 국내 다양한 수산자원 분석에 확대 적용하여 국가영양성분 데이터베이스 구축에 기여할 수 있을 것으로 사료된다. This study was performed to validate simultaneous analyses using reverse phase-high performance liquid chromatography (HPLC) with diode-array detection for vitamin B₁ (thiamin) and B₃ (nicotinic acid and nicotinamide) and HPLC-fluorescence detection for vitamin B₂ (flavin adenine dinucleotide (FAD), riboflavin 5′-monophosphate (FMN), and riboflavin) in seafood. The validated methods were applied for the analysis of B1~3 vitamers in 20 kinds of seafood (4 fish, 3 crustaceans, 3 sea algae, 3 cephalopods, 4 shellfish, and 3 others). The quantification limits (㎎/100 g) of the vitamin analyses were 0.0010 ㎎/100 g for thiamin, 0.0007∼0.0021 ㎎/100 g for FAD, FMN, and riboflavin, and 0.00001∼0.00014 ㎎/100 g for nicotinic acid and nicotinamide, with the lowest for riboflavin. Recovery of thiamin, riboflavin, nicotinic acid, and nicotinamide from standard reference materials 1849a and 3290 was 98.5∼105.8%, whereas it showed a wide range of 79.9∼120.1% for B1∼3 vitamers from seafood. Specifically, FMN was found to give the lowest (from mussel) and the highest (from Pacific herring) recoveries, which were acceptable as per official guidelines. Analyses of B1~3 vitamers showed coefficient variations of 3.1~5.4% for repeatability and 4.2∼8.0% for reproducibility, showing good precision. Total vitamin B₁, B₂, and B₃ content (㎎/100 g) of 20 types of seafood were 0.00∼0.84, 0.05∼0.47, and 0.00∼11.96, respectively. Nicotinic acid was detected in most types of seafood, whereas nicotinamide was found in only four. B₂ vitamers were observed in all seafood. The z-scores of proficiency tests for vitamin B1∼3 analyses were within ±2, indicating excellent proficiency. This study provides reliable analysis data on B₁, B₂, and B₃ vitamers of seafood, which would be used to update the national nutrient food composition table.

Riboflavin Status Influences the Biosynthesis of Flavin Peptides and Related Enzyme Activities in Rat Liver Mitochondria

신숙,김재영,박인국,Shin, Sook,Kim, Jae-Young,Park, In-Kook The Korean Society for Integrative Biology 1995 동물학회지 Vol.38 No.4

리보플라빈 결핍이 쥐간의 미토콘드리아의 플라빈 펩티드 합성, MAO, 숙신산 탈수소 효소 및 아세틸콜린 에스테라아제 활성 그리고 에피네프린가 노르에피네프린 함량에 미치는 영향을 조사하였다. 미토콘드리아내 탐지된 14C-리보플라빈의 방사선 함량과 트립신-가수분해 및 트립신-비가수분해 플라빈 펩티드의 농도의 증가는 리보플라빈 결핍시 현저히 나타났다. 미토콘드리아내 합성율은 2주째에 160% 이상으로 나타났다. MAO와 숙신산 탈수소 효소 활성은 리보플라빈 상태에 따라 현저히 감소하였으나, 아세틸콜린에스테라아제는 영향을 받지 않았다. 에피네프린과 노르에피네프린 함량도 현저히 감소하는 것으로 나타났다. 쥐간의 미토콘드리아내 플라빈 펩티드 합성, MAO, 숙신산 탈수소 효소 활성, 카테콜라민 농도는 리보플라빈 결핍상태와 특히 그 지속기간에 따라 변화하였다. The effeds of riboflavin defidency on the biosynthesis of flavin pepddes and levels of flavoenzymes and catecholamines have been investigated. The percentage of 14C. riboflavin radioactivity formed in mitochondria appeared to increase up to 2 weeks but started to decline at 3 weeks. A significant increase of radioactivity incorporation into mitochondria and into trypsin-digestable plus trypsin-non-digestibie flavin peptides was detected in riboflavin-deficient animals. More than 35% of incorporation was observed at the end of the first week and 160% higher incorporation was observed in fiavin peptide after the second week. Activities of MAO and succinate dehydrogenase were affected markedly by riboflavin status whereas those of acetyichoilnesterase were not affected. Riboflavin defidency also brought about marked reductions in levels of epineplrrine and norepinephrine. it is concluded that the levels of flavin peptides, MAO and succinate dehydrogenase, and catecholamines were affected significanily by the availability of riboflavin and in particular the duration of its depiction.

Liquid Chromatography-Tandem Mass Spectrometry Analysis of Riboflavin in Beagle Dog Plasma for Pharmacokinetic Studies

Hyeon Myeong Jeong,Beom Soo Shin,Soyoung Shin 한국질량분석학회 2020 Mass spectrometry letters Vol.11 No.1

Riboflavin is a water-soluble vitamin, which serves as a precursor to flavin mononucleotide and flavin adenine dinu-cleotide. This study aimed to develop a simple and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) analy-sis for the quantification of riboflavin in the Beagle dog plasma. This method utilized simple protein precipitation with acetonitrile and 13 C 4 , 15 N 2 -riboflavin was used as an internal standard (IS). For chromatographic separation, a hydrophilic interac-tion liquid chromatography (HILIC) column was used with gradient elution. The mobile phase consisted of 0.1% (v/v) aqueous formic acid with 10 mM ammonium formate and acetonitrile with 0.1% (v/v) formic acid. Since riboflavin is an endogenous compound, 4% bovine serum albumin in phosphate buffered saline was used as a surrogate matrix to prepare the calibration curve. The quantification limit for riboflavin in the Beagle dog plasma was 5 ng/mL. The method was fully validated for its specificity, sensitivity, accuracy and precision, recovery, and stability according to the US FDA guidance. The developed LC-MS/MS method may be useful for the in vivo pharmacokinetic studies of riboflavin.