GC Technical Note

GT086
GL Sciences Inc.

Purity Test and Fatty Acid Content Ratio Test of Polysorbate 80- Supplement II to the Japanese Pharmacopoeia, 16th Edition

A harmonized test was agreed for Polysorbate 80 in the three pharmacopoeias of Japan, Europe and theUnited States. Based on this agreement, test methods were revised in Supplement 2 of the JapanesePharmacopoeia (JP16), 16th Edition, and analysis using gas chromatography (GC) was added.
In accordance with Supplement 2 of the Japanese Pharmacopoeia, 16th Edition, a purity test and fatty acidcontent ratio test were conducted. Excellent results were obtained.

Purity (2) Ethylene oxide and 1,4-dioxane
Test sample for system suitability testuVolt
100000102010Time (min)20uVolt

20000

Standard sample

10000

203010Time (min)2010Time (min)20010000

uVolt

20000

Sample0Conditions
SystemColumn: GC - FID - HS (Tekmar HT-3): InertCap 5
0.53 mm I.D. x 50 m df = 5 μm
(Cat.No. 1010-18459)
Col. Temp.
: 70 ˚C - 10 ˚C/min – 250 ˚C (5 min)Carrier Gas: He 30 kPa
Injection: Split 3.5:185 ˚C
Injection Vol.
: Headspace Gas 1 mLDetection: FID Range 10^0250 ˚CSample: Standard
Sample heating temp. : 80 ˚C
Sample heating time 30 min
Headspace Vial
: 9.0 mLAnalyte

1. Acetaldehyde
2. Ethylene oxide
3. 1,4-Dioxane
Resolution (1, 2): 12.77 [≧ 2.0]

* JP specified conditions in []GL Sciences GC Technical Note

Test for fatty acid content ratio (system suitability)
In the “Polysorbate 80” fatty acid content ratio test, it is necessary to prepare
a mixed reagent of eight fatty acid methyl ester components. However, theconcentration specified by the test method varies for each component; thepreparation of this standard takes time and labor.
To simplify the preparation of the standard, the following is recommended;“Mixture of Fatty Acid Methyl Esters (Cat. No. 1021-58220)”diluted in nheptane according to the test method and measured using a gas
chromatograph – the mixing ratio is shown on the left.

Methyl Myristate(5 %)Methyl Palmitate(10 %)
Methyl Stearate(15 %)
Methyl Arachidate(20 %)
Methyl Oleate(20 %)
Methyl cis-11-Eicosenoate (10 %)Methyl Behenate(10 %)
Methyl Lignocerate(10 %)*One-point advice[If the weighing of the fatty acid methyl ester mixture isunsuccessful...]The Fatty Acid Methyl Ester Mixture (Cat. No. 1021-58220) is
supplied as a solid, it may be difficult to weigh in the solid state,
If this occurs, warm it in a warm water bath and return it to a liquidstate before use.
See page 4 of this Technical Note for details and prices, etc.
* A single-component sample of each component is also available. (undiluted)

uVolt
0.00E+002.00E+054.00E+056.00E+058.00E+051.00E+06[Test sample for system suitability test]○ Resolution of Methyl Stearate (3), Methyl Oleate (4)⇒ 4.21 [≧1.8]○ Number of theoretical plates of the peak of Methyl
Stearate (3)→690,723 [≧30,000 steps].

423516* Figures in parentheses indicate the conditions specified inthe JP.

7801020Time (min)3040uVolt
0 20000 40000 60000 80000100000120000

10-fold dilution○ SN ratios of Methyl Myristate(1)⇒ 229 [≧5]423

* JP specified conditions in []51010620Time (min)Conditions
SystemColumnColumn Cat.No.
Col. Temp.
Carrier Gas
Injection
Injection Vol.
Detection
Sample7830

40Analyte: GC - FID: InertCap Pure-WAX
0.32 mm I.D. x 30 m df = 0.5 μm: 1010-68244: 80 ˚C - 10 ˚C/min – 220 ˚C (40 min)
: He 110 kPa: Split 1:20250 ˚C: 1 μL: FID Range 10^0250 ˚C: Standard
Analyte in Heptane

1. Methyl Myristate
2. Methyl Palmitate
3. Methyl Stearate
4. Methyl Oleate
5. Methyl Arachidate
6. Methyl cis-11-Eicosenoate
7. Methyl Behenate
8. Methyl LignocerateGL Sciences GC Technical Note
Test for fatty acid content ratio (reference: mixed fatty acid methyl ester TS)

Methyl Myristate (5%)
Methyl Palmitate (16%)
Methyl Palmitoleate (8%)
Methyl Stearate (6%)
Methyl Oleate (58%)
Methyl Linoleate (18%)
Methyl Linolenate (4%)

For the qualitative analysis of samples, a chromatogram of"mixed fatty acid methyl ester TS" should be used. This "mixed fatty acid methyl ester TS"
should be prepared at the time of analysis because the type of fatty acid methyl ester isspecified and there is no prior indication of the mixing ratio.
In this application, a sample with the mixing ratio shown on the left was diluted with nheptane and measured using a gas chromatograph in accordance with the general test method
for "Mixed fatty acid methyl ester TS."※The mixing ratios listed on the left are with reference to the "Composition of fatty acids" of the European Pharmacopoeia (EP) and the"Acceptance Criteria" of the U.S. Pharmacopoeia (USP). In addition, the total mixing ratio was 115%, but in practice, the total mixing ratio wasconverted to 100% and the samples prepared as follows [Methyl Myristate (4%)、 Methyl Palmitate (14%)、 Methyl Palmitoleate (7%)、Methyl Stearate (5%)、 Methyl Oleate (50%)、 Methyl Linoleate (16%)、 Methyl Linolenate (3%)]

uVolt
0.00E+00 2.00E+05 4.00E+05 6.00E+05 8.00E+05

23Col. Temp.
Carrier Gas
Injection
Injection Vol.
Detection
Sample61470102030

Time (min)Conditions
SystemColumn

5: GC-4000-FID: InertCap Pure-WAX
0.32 mm I.D. x 30 m df = 0.5 μm(Cat.No. 1010-68244): 80 ℃ - 10 ℃/min - 220℃ (40 min)
: He 110 kPa: Split 1:20250 ℃: 1 μL: FID Range 10^0250 ℃: Standard
Analyte in Heptane

Analyte
1. Methyl Myristate
2. Methyl Palmitate
3. Methyl Palmitoleate
4. Methyl Stearate
5. Methyl Oleate
6. Methyl Linoleate
7. Methyl Linolenate*One-point advice[If no desired reagent is found...]Please consult GL Sciences
In addition to the procurement and arrangementof reagents, custom-made reagents are available
on customer request.

GL Sciences disclaims any and all responsibility for any injury or damage which may be caused by this data directly or indirectly.
We reserve the right to amend this information or data at any time and without any prior announcement.

GL Sciences, Inc. JapanGL Sciences B.V.

GL Sciences, Inc. USA

GL Sciences (ShangHai) Ltd.

22-1 Nishishinjuku 6-Chome
Shinjuku-ku, Tokyo,163-1130, Japan
Phone: +81-3-5323-6620
Fax: +81-3-5323-6621
Email: world@gls.co.jp
Web: www.glsciences.com

De Sleutel 95652 AS Eindhoven
The Netherlands
Phone: +31 (0)40 254 95 31
Email: info@glsciences.eu
Web: www.glsciences.eu

4733 Torrance Blvd. Suite 255
Torrance, CA 90503
Phone: 310-265-4424
Fax:310-265-4425
Email: info@glsciencesinc.comWeb: www.glsciencesinc.com

Tower B, Room 2003,
Far East International Plaza,NO,317 Xianxia Road,
Changning District.
Shanghai, China P.C. 200032
Phone: +86 (0)21-6278-2272
Email: contact@glsciences.com.cn
Web: www.glsciences.com.cn

International Distributors
Visit our Website at www.glsciences.com/distributors