Analysis of Fructose Glucose Syrup (High Fructose Syrup) According to Japanese Pharmaceutical Excipients Method (KS-801)

According to the Japanese Pharmaceutical Excipients 2018 method (PSB Notification No. 0328-1 by the Pharmaceutical Safety Bureau, MHLW, dated March 28, 2024 (New instructions)), the assay for fructose glucose syrup (high fructose syrup) is carried out with a column filled with strong acid ion-exchange resin and meets following requirements. The use of the SUGAR KS-801 column confirmed the criteria were met.


System suitability requirements

Resolution between fructose and glucose for a 10-µL injection of the standard solution: ≥ 1.5
The relative standard deviation of each fructose and glucose peak area for the six repeated analyses: ≤ 1.0

*The version at the time of the application acquisition.


Sample: 10 μL

(Standard solution)

1.Glucose 60 mg/mL

2.Fructose 84 mg/mL

Analysis of Fructose Glucose Syrup High Fructose Syrup According to Japanese Pharmaceutical Excipients Method KS 801

Column:Shodex SUGAR KS-801 (8.0 mm I.D. x 300 mm)
Flow rate:0.6 mL/min
Column temp.:80 ℃

Analysis of Glyceryl Monooleate According to USP-NF Method (KF-802)

Glyceryl oleate is a type of non-ionic surfactant. It is an emulsion stabilizer mainly used in cosmetics. According to the United States Pharmacopeia and the National Formulary (USP 43-NF 38*), the assay for glyceryl monooleate is carried out with a column (column size: 7.5 mm ID and 60 cm length) filled with L21 packing material and meets the following requirements. The use of two GPC KF-802 columns connected in series (one column size: 8.0 mm ID and 30 cm length, i.e., total of 60 cm length) confirmed the requirements were met.

System suitability requirements

Resolution between monoglyceride and diglyceride: ≥ 1.0
The relative standard deviation of monoglyceride peak area: ≤ 2.0

*The version at the time of the application acquisition.


Sample: 40 μL

(Sample solution) 40 mg/mL Glyceryl Monooleate in THF

1.Glyceryl trioleate

2.Glyceryl dioleate

3.Glyceryl monooleate



Analysis of Glyceryl Monooleate According to USP NF Method KF 802

Column:Shodex GPC KF-802 (8.0 mm I.D. x 300 mm) x 2
Flow rate:1.0 mL/min
Column temp.:40 ℃


Chitosan (SB-806M HQ)

Chitosan is a polysaccharide containing D-glucosamine and N-acetyl-D-glucosamine; it is obtained by partial deacetylation of chitin. Chitosan is used as a coating agent, a disintegrator, a binder etc. Acetic acid aqueous solution was used as the eluent, because chitosan hardly dissolves in water but easily dissolves in an organic acid aqueous solution. Chitosan has positive charge in acetic acid aqueous solution, and is adsorbed to the packing materials. Therefore, sodium nitrate was added to the eluent for reducing the ion adsorption.

Sample : 0.1 % each*, 100 µL
Chitosan (5 – 20 Pa・S)
Chitosan (50 – 100 Pa・S)
Chitosan (200 – 600 Pa・S)
*After dissolving sample with 50 mM acetic acid aq., add sodium nitrate to a final concentration of 0.3 M.



Columns      : Shodex OHpak SB-806M HQ (8.0 mm I.D. x 300 mm) x 2
Eluent       : 50 mM CH3COOH + 0.3 M NaNO3 aq.
Flow rate    : 1.0 mL/min
Detector     : RI
Column temp. : 40 °C

Analysis of Preservatives in Accordance with “Analysis Method of Food Additives in Foods” in Japan (C18M 4E)

Analysis of food additives in foods notified by the Japanese Ministry of Health, Labor and Welfare has a section for “benzoic acid, sorbic acid and their salts, and sodium dehydroacetate’” analysis. It uses an analytical column packed with octadecylsilylated silica gel (ODS column) packed in a 4.6-mm inner diameter and 50- to 250-mm length column. In this application, we analyzed nine preservatives using an ODS column, Silica C18M 4E. All preservatives analyzed were separated well. The detection was performed at two wavelengths: 230 nm and 260 nm.

Reference: Revision of the “Analysis Law for Food Additives in Foods” (Pharmaceuticals and Foods and Foods Regulations No. 0624 No. 1 dated June 24, 2021, and Pharmaceuticals and Foods and Drug Administration No. 0624 No. 1 dated June 24, 2021)” (Attachment 3)

Sample: 20 μL, 5 μg/mL each (in CH3OH/H2O=60/40)

  1. Benzoic acid
  2. Sorbic acid
  3. Dehydroacetic acid
  4. Methyl p-hydroxybenzoate
  5. Ethyl p-hydroxybenzoate
  6. Isopropyl p-hydroxybenzoate
  7. Propyl p-hydroxybenzoate
  8. Isobutyl p-hydroxybenzoate
  9. Butyl p-hydroxybenzoate

Food Additives in Foods


Column :Shodex Silica C18M 4E (4.6 mm I.D. x 250 mm)
Eluent:A); 5 mM Citrate buffer (pH4.0)/CH3CN/CH3OH=7/2/1
        B); 5 mM Citrate buffer (pH4.0)/CH3CN/CH3OH=4/2/4
        Linear gradient (High pressure);
        (B%) 0 % (0 to 12 min), 0 % to 72 % (12 to 18 min), 72 % to 100 % (18 to 25 min),  
        100 % (25 to 31 min), 100 % to 0 % (31 to 32 min), 0 % (32 to 42 min) 
Flow rate:1.0 mL/min
Detector:UV (230 nm, 260 nm)
Column temp.:40 ℃



SEC Analysis of Etelcalcetide Hydrochloride (KW-802.5)

Etelcalcetide hydrochloride is a synthetic peptide consisting of eight amino acid residues. It is used for the treatment of secondary hyperparathyroidism. Etelcalcetide was analyzed by PROTEIN KW-802.5, an aqueous SEC (GFC) column. An etelcalcetide hydrochloride-derived main peak and a peak assumed to be the sample matrix origin were observed.

Sample: 20 μL

  1. 1.Etelcalcetide hydrochloride 5 mg/mL



Column:Shodex PROTEIN KW-802.5 (8.0 mm I.D. x 300 mm)
Flow rate:0.5 mL/min
Detector:UV (225 nm)
Column temp.:25 ℃



Analysis of Epoetin According to USP-NF Method (LW-803)

According to the United States Pharmacopeia and the National Formulary (USP 41-NF 36*), analysis of high molecular proteins in “Epoetin” (erythropoietin preparation) should be carried out with a column filled with L20 packing material and meets following requirements. The PROTEIN LW-803 confirmed the requirements were met.

System suitability requirements Retention time: 8 to 10 min
Relative retention times: 0.9 (dimer) and 1.0 (monomer)
Relative standard deviation (RSD): ≤ 2.0%

*The version at the time of the application acquisition.

Sample: 40 μL
Erythropoietin 2 mg/mL (in Eluent)
Heat at 80 ℃ for 30 min

  1. Dimer
  2. Monomer
Chromatogram of indigestible dextrin
ColumnShodex PROTEIN LW-803 (8.0 mm I.D. x 300 mm)
Eluent20 mM Sodium citrate + 100 mM NaCl aq. (pH6.9 adjust with Hydrochloric acid)
Flow rate1.0 mL/min
DetectorUV (230 nm)
Column temp.25 ℃

Freshness Index of Fish Flesh (K Value)

Decomposition of ATP is one of the major biochemical postmorten changes in muscles of fish and shellfish. During this process, ATP, ADP, and AMP decompose quickly to form and build-up inosine and hypoxanthine. The increase in the ratio of inosine and hypoxanthine to the total quantity of ATP and its related substances corresponds to the decline of the fish’s freshness well. This ratio is called K value and used as an index to represent the freshness of fish flesh.


Sample : ATP, ADP, AMP, IMP, Hypoxanthine, Inosine

Analysis of Anthocyanidins (C18M 4D)

Anthocyanidins are polyphenols found in grape skins and blueberries. They have antioxidant and anti-inflammatory effects, which are expected to improve vision and nerve health. In this application, we performed separation of six major anthocyanidins using Silica C18 M 4D, a silica-based reversed-phase chromatography column. Six anthocyanidins were analyzed within 20 minutes using an isocratic elution.

Sample: 10 μL

  1. Delphinidin 20 μg/mL
  2. Cyanidin 10 μg/mL
  3. Petunidin 40 μg/mL
  4. Pelargonidin 10 μg/mL
  5. Peonidin 20 μg/mL
  6. Malvidin 20 μg/mL

Dissolved in 1 % TFA/CH3OH=30/70

chromatogram of Anthocyanidins
ColumnShodex Silica C18M 4D (4.6 mm I.D. x 150 mm)
Eluent5 % HCOOH/CH3CN/CH3OH=80/10/10
Flow rate1.0 mL/min
DetectorVIS (520 nm)
Column temp.40 ℃

Analysis of Perchloric Acid by LC/MS (SI-35 2B)

IC SI-35 2B, an anion analysis column was used to analyze perchlorate and its related anions. LC/MS analysis of perchlorate ions has gained attention in recent years, which includes a published EPA Method 6850. Perchlorate ions can be detected at m/z 99 and m/z 101, and their peak area ratio corresponds to the isotopic ratio of 35Cl and 37Cl. Fragment ions of perchlorate ions, m/z 83 and m/z 85 are detected. Their m/z are the same as chlorate ion m/z, but since perchlorate ions and chlorate ions have sufficiently different retention times, this allows their differentiation. Additionally, the m/z 99 ion of perchlorate ions may be influenced by H34SO4, which has the same m/z, but as they also have distinct retention times enabling the differentiation.
After confirming the retention times of each ion using a mixed standard solution (1 μg/mL each), we measured a mixed standard solution containing 50 ng/mL each. Peaks of perchlorate ions were observed at both m/z 99 and m/z 101. The effects of other components on the perchlorate ion peaks can be checked by the peak area ratio of 35ClO4 to 37ClO4. In this analysis, even at low concentrations of perchlorate ions, peaks derived from 37ClO4 can be determined.

Sample: 5 μL
Mixed standard solution (1 μg/mL each)

  1. Perclorate, ClO4
  2. Hydrogen sulfate, HSO4
  3. Chlorate, ClO3
  4. Chloride, Cl
chromatogram of perclorate

Sample: 5 μL
Mixed standard solution (50 ng/mL each)

  1. Perclorate, ClO4
chromatogram of perclorate
ColumnShodex IC SI-35 2B (2.0 mm I.D. × 50 mm)
Eluent(A); 100 mM NH4HCO3 aq., (B); CH3CN, (C); H2O
gradient; 30 % (A), 10 % (B), 60 % (C) (0 min) to 80 % (A), 10 % (B), 10 % (C) (10 to 16 min) to 30 % (A), 10 % (B), 60 % (C) (16.5 to 20 min)
Flow rate0.25 mL/min
DetectorESI-MS (negative, SIM)
Column temp.45 ℃

Rapid Analysis of Trivalent Chromium and Hexavalent Chromium (VN-50 4D)

HILICpak VN-50 4D, a hydrophilic interaction chromatography (HILIC) column modified with diol functional group was coupled with an ICPMS for the separation analysis of trivalent chromium (Cr(III)) and hexavalent chromium (Cr(VI)). In this application, the eluent used has a high proportion of aqueous solution containing 2,6-pyridinedicarboxylic acid (PDCA), also known as dipicolinic acid. The presence of PDCA in the eluent leads Cr(III) to form a stable (Cr(III)-PDCA) complex. The chromatogram below compares the elution profiles with different acetonitrile (ACN) concentrations, ranging from 2 % to 4 %. It demonstrates that Cr(VI) elutes before Cr(III)-PDCA under these conditions (The upper limit of ACN was set at 4 %, as higher ACN concentrations lead to decreased sensitivity). Although simultaneous separation of Cr(VI) and Cr(III)-PDCA is achievable under all eluent conditions, increased ACN concentration promoted earlier elution of Cr(III)-PDCA and also improved their peak shapes. Therefore, 4 % ACN is an optimal concentration for this analysis. In chromium analysis using ICPMS, interference from chloride ions is a concern. The ability to separate chloride ions from chromium prior to introduction into the ICP-MS is crucial for minimizing its impact on the quantification.
*The HPLC system used in this analysis is biocompatible, and the housing material for VN-50 4D is PEEK.

Sample: 100 μL
25 μg L-1 Cr(Ⅵ)-Cr(Ⅲ)PDCA solution and 0.001 mol L-1 HCl

  1. light grey:2 % ACN
  2. grey:3 % ACN
  3. black:4 % ACN
chromatogram of Cr

Data Provided by Prof. Masaharu Tanimizu, Department of Applied Chemistry for Environment, School of Science and Technology, Kwansei Gakuin University

Ito, A., Morishita, Y., Morimoto, T., Tanimizu, M. Rapid determination of chromium species in environmental waters using a diol-bonded polymer-stationary column under water-rich conditions coupled with ICPMS. ANAL. SCI. (2024).

ColumnShodex HILICpak VN-50 4D (4.6 mm I.D. × 150 mm)
Eluent50 mmol L-1 CH3COONH4, 2 mmol L-1 PDCA, 2% ACN, pH7.0 ± 0.2
50 mmol L-1 CH3COONH4, 2 mmol L-1 PDCA, 3% ACN, pH7.0 ± 0.2
50 mmol L-1 CH3COONH4, 2 mmol L-1 PDCA, 4% ACN, pH7.0 ± 0.2
Flow rate0.6 mL min-1
Column temp.Room temperature