According to the United States Pharmacopeia and the National Formulary Pharmacopeial Forum (PF 48(4)*), mannitol assay should be carried out with an HPLC and a column filled with L22 packing material, and meets following requirements. The use of SUGAR SP0810, a sugar analysis column, confirmed the requirements were met.
System suitability requirements:
Tailing factor: ≤ 2.0
Relative standard deviation (RSD): ≤ 2.0%
*The version at the time of the application acquisition.

Sample : 80 μL
1. USP Mannitol RS 2.5 mg/mL
Column : Shodex SUGAR SP0810 (8.0 mm I.D. x 300 mm)
Eluent : H2O
Flow rate : 1.0 mL/min
Detector : RI
Column temp. : 80 ℃
According to the United States Pharmacopeia and the National Formulary (USP-NF 2022 ISSUE 3* Effective December 1, 2022), analysis of contaminants sucrose and sorbitol in cranberry juice should be carried out with a column with L19 packing material and meets following requirements. The EP SC1011-7F confirmed the requirements were met.
System suitability requirements:
Resolution of sucrose and sorbitol: ≥ 1.8
Relative standard deviation (RSD): ≤ 2.0 %
*The version at the time of the application acquisition.

Sample :
0.1 mg/mL each, 20 μL
1. Sucrose
2. Sorbitol
Columns : Shodex EP SC1011-7F (7.8 mm I.D. x 300 mm)
Eluent : H2O
Flow rate : 0.5 mL/min
Detector : RI
Column temp. : 85 ℃
HILICpak VN-50 4D, a polymer-based HILIC mode column, was used to analyze and successfully separate three analytes and their phosphorylated forms. VN-50 4D retains polar substances, which are not retained well by regular reversed-phase mode ODS columns. Thus, it is suitable for the analysis of sugars, sugar intermediates, and phosphorylated compounds with high polarity. Analytes studied were Glucose, D-Ribose, Pyruvate, and their respective phosphorylated compounds. From the chromatograms, we see the non-phosphorylated analytes elute first on the VN-50 4D, while the phosphorylated analytes eluate later due to them being higher charged species. For the pyruvate and phosphoenolpyruvate analysis, we want to note that pyruvate did not retain on the column too long because of its low charged state. Lastly, the asterisks shown are phosphate impurities found in the phosphorylated samples.

Column : Shodex HILICpak VN-50 4D (4.6mmI.D. x 150mm each)
Eluent : 70% Acetonitrile, 30% 50mM Ammonium Bicarbonate
Flow rate : 1 mL/min
Detector : Shodex RI
Column temp. : 30°C
Ascorbic acid and isoascorbic acid were separated using two columns of RSpak DE-413 (a column for polymer-based reversed phase chromatography). The calibration curve of ascorbic acid and isoascorbic acid shows good linearity respectively in the range from 1 to 100 μg/mL.
Sample : 10 μL
5 μg/mL each (in 0.1 % metaphosphoric acid)
1. Ascorbic acid
2. Isoascorbic acid


Column : Shodex RSpak DE-413 (4.6 mm I.D. x 150 mm) x 2
Eluent : 10 mM H3PO4 aq.
Flow rate : 0.6 mL/min
Detector : UV (254 nm)
Column temp. : 40 ℃
In R&D and QC of nucleic acid drugs such as antisense nucleic acids require development of analytical methods that separate the target synthetic oligonucleotide from its impurities as much as possible. In this application, four compounds were analyzed using HILICpak VN-50 2D, a polymer-based HILIC column. The four compounds were 20mer synthetic oligo-DNAs: one with the target base sequence and its three analogs with 1 to 3 base alterations. By having different bases make their hydrophilicities different from each other. Four synthetic oligo-DNAs were separated by HILIC mode using this hydrophilicity differences. The application developed here does not require a use of ion-pairing reagent nor highly concentrated salt in the eluent. Therefore, it is suitable for LC/MS analysis of oligonucleotides.
Sample : Synthesized oligo-DNAs (crude),1 μL
1. 20mer, ATACCGATTAAGCGAAGTTT
2. 20mer, ATACCGATTAAGCGAATTTT
3. 20mer, ATACCGATTAAGCTAATTTT
4. 20mer, ATACCGATTAATCTAATTTT

Column : Shodex HILICpak VN-50 2D (2.0 mm I.D. x 150 mm)
Eluent : (A)50 mM HCOONH4 aq. /(B) CH3CN
Linear gradient ;
(B %) 62 to 56 % (0 to 10 min), 56 % (10 to 20 min),
56 to 62 % (20 to 20.01 min), 62 % (20.01 to 25 min)
Flow rate : 0.2 mL/min
Detector : UV (260 nm) (small cell volume), ESI-MS (SIM Negative)
Column temp. : 60 ℃
In R&D and QC of nucleic acid drugs such as antisense nucleic acids require development of analytical methods that separate the target synthetic oligonucleotide from its impurities as much as possible. In this application, four compounds were analyzed using HILICpak VN-50 2D, a polymer-based HILIC column. The four compounds were the target synthetic oligo-DNA (20mer; n nucleotides) and its three analogs with 1 to 3 truncated nucleotides (19mer, 18mer, and 17mer; n-1, n-2, and n-3 nucleotides respectively). Under HILIC mode, the higher the hydrophilicity, the stronger the retention becomes. Thus, the four oligo-DNAs eluted in the order of smaller to larger oligomer. The application developed here does not require a use of ion-pairing reagent nor highly concentrated salt in the eluent. Therefore, it is suitable for LC/MS analysis of oligonucleotides.
Sample : Synthesized oligo-DNAs (crude), 1 μL
1. 20mer (n), CTTCTCATGGTTCTTCGGAA
2. 19mer (n-1), TTCTCATGGTTCTTCGGAA
3. 18mer (n-2), TCTCATGGTTCTTCGGAA
4. 17mer (n-3), CTCATGGTTCTTCGGAA

Column : Shodex HILICpak VN-50 2D (2.0 mm I.D. x 150 mm)
Eluent : (A)50 mM HCOONH4 aq. /(B) CH3CN
Linear gradient ;
(B %) 62 to 56 % (0 to 10 min), 56 % (10 to 20 min),
56 to 62 % (20 to 20.01 min), 62 % (20.01 to 25 min)
Flow rate : 0.2 mL/min
Detector : UV (260 nm) (small cell volume), ESI-MS (SIM Negative)
Column temp. : 60 ℃
Polystyrene standards were analyzed using GPC HK-401, an organic SEC (GPC) column for ultra-rapid analysis. The RI chromatograms showed that the benzene peak overlapped with the eluent-related negative peak.
RI
Sample : 0.5 % each, 5 µL
1. Polystyrene (Mp: 1,270)
2. Polystyrene (Mp: 580)
3. n-Propylbenzene (MW: 120)
4. Benzene (MW: 78)
UV
Sample : 0.1 % each, 5 µL
1. Polystyrene (Mp: 1,270)
2. Polystyrene (Mp: 580)
3. n-Propylbenzene (MW: 120)
4. Benzene (MW: 78)

Columns : Shodex GPC HK-401 (4.6 mm I.D. x 150 mm) x 2
Eluent : THF
Flow rate : 1.0 mL/min
Detector : RI (small cell volume), UV (254 nm) (small cell volume)
Column temp. : 25 °C