2.4.28. 2-Ethylhexanoic acid
EUROPEAN PHARMACOPOEIA 7.0
of potassium solution to stand at room potassium iodide R . Allow the test solution temperatur temperature e for about 50 min or at 70 °C for about 4 min.
with the following temperature programme: Time (min) 0-2
Acid reagent . Heavy metal-free hydrochloric acid R. solution n of sodium Reducing reagent . A 6 g/L solutio sodium tetrahydroborate tetrahydroborate R in a 5 g/L solution of sodium hydroxide R.
Column
2 - 7.3
The instrumental parameters in Table 2.4.27.-2 may be used.
7.3 - 10.3
Temperature (°C) 40 40
→
200
200
Mercury
Injection port
200
Sample solution. Test solution or blank solution, as prescribed above.
Detector
300
Rate (°C/min) –
isothermal
30
linear gradient
–
isothermal
Comment
Inject Inject 1 μL of thetest sol solut utio ion n and 1 μ L of the refere reference nce solution. solution.
Acid reagent . A 515 g/L solution of heavy metal-free hydrochloric acid R. Reducing reagent . A 10 g/L solution of stannous stannous chloride R in dilute heavy metal-free hydrochloric acid R. The instrumental parameters in Table 2.4.27.-2 may be used.
The test is not valid unless the resolution resolution between between the peaks corresponding to 2-ethylhexanoic acid (first peak) and the internal internal standard is at least 2.0. Calculate the percentage content of 2-ethylhexanoic acid from the expression expression :
Table 2.4.27.-2 As
Hg
19 193.7
253.7
Wavelength
nm
Slit width
nm
0.2
0.5
Lamp current
mA
10
4
mL/min
1.0
1.0
mL/min
1.0
1.0
m L/min mL
7.0
7.0
Quartz (heated)
Quartz (unheated)
off
off
0.1
0.1
Acid reagent flow rate
Reducing reagent flow rate
Sample solution flow rate
Absorption cell Background corrector Nitrogen flow rate
L/min
AT
=
area of the peak peak corresponding to 2-ethylhexanoic acid in the chromatogram chromatogram obtained with the test solution,
A R
=
I T
=
area of the peak peak corresponding to 2-ethylhexanoic acid in the chromatogram obtained with the reference solution, area of the peak peak corresponding corresponding to the internal internal standard in the chromatogram obtained with the test solution,
I R
=
area of the peak peak corresponding corresponding to the internal internal standard in the chromatogram obtained with the reference solution,
mT
=
mass of the substance substance to be examined examined in the test solution, solution, in grams,
m R
=
mass of 2-ethylhexanoic acid in the reference reference solution, solution, in grams.
01/2008:20428 07/2010:20429
2.4.28. 2-ETHYLHEXANOIC ACID 2.4.29. COMPOSITION OF FATTY ACIDS IN OILS RICH IN OMEGA-3 ACIDS
Examine by gas chromatograph chromatographyy ( 2.2.28 ), ), using 3-cyclohexylpropionic acid R as the internal standard. Dissolve 100 mg of of Internal standard solution. Dissolve 3-cyclohexylpropionic acid R in cyclohexane R and dilute to 100 mL with the same solvent.
Test solution . To 0.300 g of the substance to be examined, add 4.0 mL of a 33 per cent V/V solution solution of hydrochloric hydrochloric acid R . Shake vigorously for 1 min with 1.0 mL of the internal standard solution. Allow the phases to separate (if necessary, centrifuge for a better separation). Use the upper layer. Dissolve 75.0 mg of 2-ethylhexanoic Reference solution. Dissolve 2-ethylhexanoic acid R in the internal standard solution solution and dilute dilute to 50.0 mL with the same solution. solution. To 1.0 mL of the solution add 4.0 mL of a 33 per cent V/V solution of hydrochloric acid R . Shake vigorously for V/V solution 1 min. Allow the phases to separate (if necessary, centrifuge for a better separation). separation). Use the upper layer. layer.
The assay may be used for quantitative determination of the EPA and DHA content in omega-3-containing products of fish oil in different concentrations. The method is applicable to triglycerides or ethyl esters and the results are expressed as triglycerides or ethyl esters, respectively. EPA AND DHA Gas chromatography ( 2.2.28 ). ). Carry out the operations as rapidly as possible, avoiding exposure to actinic light, oxidising agents, oxidation catalysts (for example, copper and iron) and air. The assay is carried out on the methyl or ethyl esters of (all- Z )-eicosa-5,8,11,14,17-pentaenoic acid (EPA; 20:5 n-3) and Z )-eicosa-5,8,11,14,17-pentaenoic (all- Z )-docosa-4,7,10,13,16,19-hexaenoic acid (DHA; 22:6 n-3) in Z )-docosa-4,7,10,13,16,19-hexaenoic the substance to be examined.
The chromatographic procedure may be carried out using:
Internal stand ard . Methyl tricosanoate R .
— a wide-bore wide-bore fused-silica fused-silica column column 10 m long and 0.53 mm in internal internal diameter diameter coated coated with macrogol 20 000 2-nitroterephthalate R (film thickness 1.0 μ m),
Test solution (a)
— helium for chromatography chromatography R as the carrier gas at a flow rate of 10 mL/min, — a flame-ionisat flame-ionisation ion detecto detector, r,
130
A. Dissolve the mass of sample to be examined according to Table 2.4.29.-1 2.4.29.-1 and about 70.0 mg of the internal internal standard in a 50 mg/L solution of butylhydroxytoluene R in trimethylpentane R and dilute to 10.0 mL with the same solution. Gentle heating (up to 60 °C) may be applied to dissolve the internal standard.
See the information section on general monographs (cover pages)
2.4.29. Composition of fatty acids in oils rich in omega-3 acids
EUROPEAN PHARMACOPOEIA 7.0
Table 2.4.29.-1. Approximate sum EPA + DHA (per cent)
Mass of sample to be examined (g)
30 - 50
0.4 - 0.5
50 - 70
0.3
70 - 90
Split ratio : 1:200, alternatively splitless with temperature control (sample solutions need to be diluted 1/200 with a 50 mg/L solution of butylhydroxytoluene R in trimethylpentane R before injection). Temperature :
0.25
Ethyl esters are now ready for analysis. For triglycerides continue as described in step B.
Column
Time (min) 0-2 2 - 25.7
Temperature (°C) 170 170
→
240
B. Introduce 2.0 mL of the solution obtained in step A into a 240 25.7 - 28 quartz tube and evaporate the solvent with a gentle current Injection port 250 of nitrogen R. Add 1.5 mL of a 20 g/L solution of sodium hydroxide R in methanol R, cover with nitrogen R, cap Detector 270 tightly with a polytetrafluoroethylene-lined cap, mix and heat on a water-bath for 7 min. Allow to cool. Add 2 mL of boron Detection : flame ionisation. trichloride-methanol solution R , cover with nitrogen R, cap Injection : 1 μ L, twice. tightly, mix and heat on a water-bath for 30 min. Cool to System suitability : 40-50 °C, add 1 mL of trimethylpentane R, cap and shake vigorously for at least 30 s. Immediately add 5 mL of a — in the chromatogram obtained with reference solution (b), saturated sodium chloride solution R, cover with nitrogen R, the area per cent composition increases in the following cap and shake thoroughly for at least 15 s. Transfer the order: methyl palmitate, methyl stearate, methyl arachidate, upper layer to a separate tube. Shake the methanol layer methyl behenate; the difference between the percentage once more with 1 mL of trimethylpentane R. Wash the area of methyl palmitate and that of methyl behenate is less combined trimethylpentane extracts with 2 quantities, than 2.0 area per cent units; each of 1 mL, of water R and dry over anhydrous sodium — resolution : minimum of 1.2 between the peaks due to sulfate R . Prepare 3 solutions for each sample. docosahexaenoic acid methyl ester and to tetracos-15-enoic Test solution (b) . Dissolve 0.300 g of the sample to be acid methyl ester in the chromatogram obtained with examined in a 50 mg/L solution of butylhydroxytoluene R reference solution (c); in trimethylpentane R and dilute to 10.0 mL with the same — in the chromatogram obtained withtest solution (a), the peaks solution. Proceed as described for test solution (a). due to methyl tricosanoate and any heneicosapentaenoic acid Reference solution (a1 ). Dissolve about 70.0 mg of the methyl ester or ethyl ester (C21:5) present when compared internal standard and 90.0 mg of eicosapentaenoic acid ethyl with the chromatogram obtained with test solution (b) are ester CRS in a 50 mg/L solution of butylhydroxytoluene R clearly separated (if not, a correction factor has to be used). in trimethylpentane R and dilute to 10.0 mL with the same Calculate the percentage content of EPA and DHA using the solution. Gentle heating (up to 60 °C) may be applied to following expression and taking into account the assigned value dissolve the internal standard. of the reference substances: Reference solution (a 2 ). Dissolve 60.0 mg of docosahexaenoic acid ethyl ester CRS and about 70.0 mg of the internal standard in a 50 mg/L solution of butylhydroxytoluene R in trimethylpentane R and dilute to 10.0 mL with the same solution. Gentle heating (up to 60 °C) may be applied to m1 = mass of the internal standard in test solution (a), dissolve the internal standard. in milligrams; For both reference solution (a 1) and reference solution (a 2) m2 = mass of the sample to be examined in test proceed as described for test solution (a) step A when analysing solution (a), in milligrams; ethyl esters. For analysis of triglycerides, continue with step B in the same manner as for test solution (a) and prepare m x ,3 = mass of the internal standard in reference 3 solutions for each sample. solution (a1) (EPA determination), or in reference solution (a 2) (DHA determination), in milligrams; Reference solution (b). Into a 10 mL volumetric flask dissolve 0.3 g of methyl arachidate R, 0 . 3 g o f m ethyl behenate R, 0 . 3 g o f m x,r = mass of eicosapentaenoic acid ethyl ester CRS methyl palmitate R and 0.3 g of methyl stearate R in a 50 mg/L in reference solution (a 1) or docosahexaenoic solution of butylhydroxytoluene R in trimethylpentane R and acid ethyl ester CRS in reference solution (a 2), in dilute to 10.0 mL with the same solution. milligrams; Reference solution (c). Into a 10 mL volumetric f lask dissolve A x = area of the peak due to eicosapentaenoic acid ester a sample containing about 55.0 mg of docosahexaenoic acid or docosahexaenoic acid ester in the chromatogram methyl ester R and about 5.0 mg of tetracos-15-enoic acid obtained with test solution (a); methyl ester R in a 50 mg/L solution of butylhydroxytoluene R in trimethylpentane R and dilute to 10.0 mL with the same A x,r = area of the peak due to eicosapentaenoic acid solution. ester in the chromatogram obtained with reference Column : solution (a1) or to docosahexaenoic acid ester in the chromatogram obtained with reference solution (a 2) ; — material : fused silica; — dimensions : l = at least 25 m, Ø = 0.25 mm ; — stationary phase : bonded macrogol 20 000 R (film thickness 0.2 μ m).
Carrier gas : hydrogen for chromatography R or helium for chromatography R. Flow rate : 1 mL/min.
General Notices (1) apply to all monographs and other texts
A1
=
area of the peak due to the internal standard in the chromatogram obtained with test solution (a) ;
A x ,3
=
area of the peak due to the internal standard in the chromatogram obtained with reference solution (a 1) (EPA determination) or with reference solution (a 2) (DHA determination);
131
2.4.30. Ethylene glycol and diethylene glycol in ethoxylated substances
C
=
conversion factor between ethyl ester and triglycerides,
C = 1.00 for ethyl esters,
EUROPEAN PHARMACOPOEIA 7.0
Temperature :
Column
Time (min) 0 - 40
C = 0.954 for EPA,
40 - 45
C = 0.957 for DHA.
45 - 65
TOTAL OMEGA-3 ACIDS
Temperature (°C) 80 → 200 200
→
230
230
Injection port
250
Detector
250
From the assay for EPA and DHA, calculate the percentage content of the total omega-3 acids using the following expression Detection : flame ionisation. and identifying the peaks from the chromatograms: Injection : 2 μL. Relative retention with reference to 1,2-pentanediol (retention time = about 19 min): ethylene glycol = about 0.7; diethylene glycol = about 1.3.
EPA =
percentage content of EPA ;
DHA =
percentage content of DHA ;
An-3
sum of the areas of the peaks due to C18:3 n-3, C18:4 n-3, C20:4 n-3, C21:5 n-3 and C22:5 n-3 esters in the chromatogram obtained with test solution (b); area of the peak due to EPA ester in the chromatogram obtained with test solution (b); area of the peak due to DHA ester in the chromatogram obtained with test solution (b).
=
01/2008:20431
2.4.31. NICKEL IN HYDROGENATED VEGETABLE OILS
Atomic absorption spectrometry ( 2.2.23, Method I ). CAUTION: when using closed high-pressure digestion vessels and microwave laboratory ovens, be familiar with the safety ADHA = and operating instructions given by the manufacturer. The reagents magnesium nitrate R and ammonium dihydrogen phosphate R must be controlled for nickel before use. The actual nickel content is taken into account in the calculation of the nickel content of the sample. 01/2008:20430 Test solution . Weigh 0.250 g ( m) of the substance to be examined into a suitable high-pressure-resistant digestion vessel (fluoropolymer or quartz glass), add 6.0 mL of nickel-free nitric 2.4.30. ETHYLENE GLYCOL acid R and 2.0 mL of strong hydrogen peroxide solution R . AND DIETHYLENE GLYCOL IN Prepare a blank solution in the same manner. Place the ETHOXYLATED SUBSTANCES closed vessels in a laboratory microwave oven and digest with an appropriate programme, e.g. 1000 W for 40 min. Allow Ethoxylated substances may contain varied amounts the digestion vessels to cool before opening. Add 2.0 mL of of ethylene glycol and diethylene glycol, as a result strong hydrogen peroxide solution R and repeat the digestion of the manufacturing process. The following method step. Allow the digestion vessels to cool before opening. may be used for the quantitative determination of these Quantitatively transfer to a 25 mL flask, add 0.5 mL of a 10 g/L substances, in particular in the case of the following solution of magnesium nitrate R and 0.5 mL of a 100 g/L surfactants: macrogolglycerol ricinoleate, macrogolglycerol solution of ammonium dihydrogen phosphate R , dilute to hydroxystearate, macrogol 15 hydroxystearate, nonoxinol 9 25.0 mL with water for chromatography R and mix. and macrogol cetostearyl ether. Reference solutions . Into 4 volumetric flasks, introduce 25 μ L, 50 μ L, 75 μ L and 100 μ L of nickel standard solution Gas chromatography ( 2.2.28 ). (5 ppm Ni) R. To each flask, add 0.5 mL of a 10 g/L solution Internal standard solution. Dissolve 30.0 mg of of magnesium nitrate R, 0.5 mL of a 100 g/L solution 1,2-pentanediol R in acetone R and dilute to 30.0 mL with the of ammonium dihydrogen phosphate R and 6.0 mL of same solvent. Dilute 1.0 mL of this solution to 20.0 mL with nickel-free nitric acid R and dilute to 25.0 mL with water acetone R. for chromatography R . Mix to obtain reference solutions Test solution . Dissolve 0.500 g of the substance to be examined containing respectively 5 ng/mL, 10 ng/mL, 15 ng/mL and 20 ng/mL (ppb) of nickel. in the internal standard solution and dilute to 10.0 mL with Zero solution . In a volumetric flask, introduce 1.0 mL of a the same solution. 10 g/L solution of magnesium nitrate R, 1.0 mL of a 100 g/L Reference solution (a). Mix 30.0 mg of ethylene glycol R with acetone R and dilute to 100.0 mL with the same solvent. Dilute solution of ammonium dihydrogen phosphate R and 12.0 mL of nickel-free nitric acid R . Dilute to 50.0 mL with water for 1.0 mL to 10.0 mL with the internal standard solution. chromatography R and mix. Reference solution (b). Prepare a solution of diethylene Method . Determine the absorbance of each solution at glycol R with a concentration corresponding to the prescribed 232.0 nm using a suitable graphite furnace atomic absorption limit and using the same solvents as for the preparation of spectrometer equipped with a background compensation reference solution (a). system, a pyrolytically-coated tube, and a nickel hollow-cathode Column : lamp. Maintain the drying temperature of the furnace at 120 °C for 35 s after a 5 s ramp, the ashing temperature at 1100 °C for — material : fused silica, 10 s after a 30 s ramp, the cooling temperature at 800 °C for 5 s — size : l = 30 m, Ø = 0.53 mm, after a 5 s decrease, and the atomisation temperature at 2600 °C — stationary phase : macrogol 20 000 R (film thickness 1 μ m). for 7 s. Use the zero solution to set the instrument to zero. Using the calibration curve, determine the concentrations of Carrier gas : helium for chromatography R . the test solution and the blank solution from the corresponding Flow rate : 10 mL/min. absorptions. If necessary, dilute with the zero solution to obtain a reading within the calibrated absorbance range. Split ratio : 1:3.
AEPA
132
=
See the information section on general monographs (cover pages)
