LEMON OIL Limonis aetheroleum

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					EUROPEAN PHARMACOPOEIA 5.0                                                                                                    Lemon oil



Elution order : order indicated in the composition of           Limits : calculate the percentage content of the specified
reference solution (a). Record the retention times of these     (S)-enantiomers from the expression :
substances.
System suitability : reference solution (a) :
— resolution : minimum 1.4 between the peaks due to
    terpinen-4-ol and lavandulyl acetate.                       AS    =    area of the peak due to the corresponding
Using the retention times determined from the chromatogram                 (S)-enantiomer,
obtained with reference solution (a), locate the components     AR    =    area of the peak due to the corresponding
of reference solution (a) in the chromatogram obtained with                (R)-enantiomer.
the test solution.
                                                                — (S)-linalol : maximum 12 per cent,
Determine the percentage content of each of these               — (S)-linalyl acetate : maximum 1 per cent.
components. The percentages are within the following
ranges :                                                        STORAGE
— limonene : less than 1.0 per cent,                            In a well-filled, airtight container, protected from light, at a
— cineole : less than 2.5 per cent,                             temperature not exceeding 25 °C.
— 3-octanone : 0.1 per cent to 2.5 per cent,
                                                                                                                       01/2005:0620
— camphor : less than 1.2 per cent,                                                                                        corrected
— linalol : 20.0 per cent to 45.0 per cent,
— linalyl acetate : 25.0 per cent to 46.0 per cent,
                                                                                          LEMON OIL
— terpinen-4-ol: 0.1 per cent to 6.0 per cent,                                     Limonis aetheroleum
— lavandulyl acetate : more than 0.2 per cent,
                                                                DEFINITION
— lavandulol : more than 0.1 per cent,                          Essential oil obtained by suitable mechanical means, without
— α-terpineol: less than 2.0 per cent.                          the aid of heat, from the fresh peel of Citrus limon (L.)
— disregard limit: area of the peak in the chromatogram         Burman fil.
    obtained with reference solution (b) (0.05 per cent).       CHARACTERS
Chiral purity. Gas chromatography (2.2.28).                     Appearance : clear, mobile, pale yellow to greenish-yellow
Test solution. Dissolve 0.02 g of the substance to be           liquid with a characteristic odour. It may become cloudy at
examined in pentane R and dilute to 10 ml with the same         low temperatures.
solvent.                                                        IDENTIFICATION
Reference solution. Dissolve 10 µl of linalol R, add 10 µl of   First identification : B.
linalyl acetate R and 5 mg of borneol R in pentane R and        Second identification : A.
dilute to 10 ml with the same solvent.
                                                                A. Thin-layer chromatography (2.2.27).
Column :                                                           Test solution. Mix 1 ml of the substance to be examined
— material : fused silica,                                         in 1 ml of toluene R.
— size : l = 25 m, Ø = 0.25 mm,                                    Reference solution. Dissolve 10 mg of citropten R and
                                                                   50 µl of citral R in toluene R and dilute to 10 ml with
— stationary phase: modified β-cyclodextrin for chiral             the same solvent.
    chromatography R (film thickness 0.25 µm).
                                                                   Plate : TLC silica gel GF254 plate R.
Carrier gas : helium for chromatography R.                         Mobile phase : ethyl acetate R, toluene R (15:85 V/V).
Flow rate : 1.3 ml/min.                                            Application : 10 µl, as bands.
Split ratio : 1 :30.                                               Development : over a path of 15 cm.
Temperature :                                                      Drying : in air.
                                                                   Detection A : examine in ultraviolet light at 254 nm.
                       Time                Temperature
                       (min)                  (°C)
                                                                   Results A : see below the sequence of the zones present in
                                                                   the chromatograms obtained with the reference solution
 Column                0 - 65               50 → 180
                                                                   and the test solution.
 Injection port                                230
                                                                                                Top of the plate
 Detector                                      230
                                                                                                         A quenching zone (bergamotin)
Detection : flame ionisation.                                        Citral: a quenching zone            A quenching zone (citral)
Injection : 1 µl.                                                                                        A dark blue zone (5-geranyloxy-
                                                                                                         7-methoxycoumarin)
System suitability : reference solution :
                                                                     Citropten : a light blue            A light blue fluorescent zone
— resolution : minimum 5.5 between the peaks due to                  fluorescent zone                    (citropten)
   (R)-linalol (1st peak) and (S)-linalol (2nd peak), minimum                                            A quenching zone (psoralen
   2.9 between the peaks due to (S)-linalol and borneol                                                  derivative)
   (3rd peak) and minimum 2.7 between the peaks due                                                      A quenching zone (biakangelicin)
   to (R)-linalyl acetate (4th peak) and (S)-linalyl acetate
                                                                           Reference solution                      Test solution
   (5th peak).


General Notices (1) apply to all monographs and other texts                                                                          1895
Lemon oil                                                                                            EUROPEAN PHARMACOPOEIA 5.0



   Detection B : examine in ultraviolet light at 365 nm.                    400 nm. Plot a curve representing the absorption spectrum
   Results B : see below the sequence of the zones present in               with the absorbances as ordinates and the wavelengths as
   the chromatograms obtained with the reference solution                   abscissae. Draw as a baseline the tangent between A and B
   and the test solution.                                                   (Figure 0620.-1). The absorption maximum C is situated at
                                                                            315 ± 3 nm. From C draw a line perpendicular to the axis of
                                Top of the plate                            abscissae and intersecting AB at D. Deduct the absorbance
                                         A yellow fluorescent zone          corresponding to point D from that corresponding to point C.
                                         (bergamotin)                       The value C − D is 0.20 to 0.96 and for Italian-type lemon oil
    Citral: a quenching zone             A quenching zone (citral)          it is not less than 0.45.
                                         A bright blue fluorescent          Fatty oils and resinified essential oils (2.8.7). It complies
                                         zone (5-geranyloxy-7-              with the test for fatty oils and resinified essential oils.
                                         methoxycoumarin)
                                                                            Chromatographic profile. Gas chromatography (2.2.28) :
    Citropten : a bright blue            A bright violet-blue fluorescent   use the normalisation procedure.
    fluorescent zone                     zone (citropten)
                                                                            Test solution. The substance to be examined.
                                         A yellow fluorescent zone
                                         (psoralen derivative)              Reference solution. Dissolve 20 µl of β-pinene R, 10 µl of
                                         An orange zone (biakangelicin)     sabinene R, 100 µl of limonene R, 10 µl of γ-terpinene R, 5 µl
                                                                            of β-caryophyllene R, 20 µl of citral R, 5 µl of α-terpineol R,
          Reference solution                       Test solution            5 µl of neryl acetate R and 5 µl of geranyl acetate R in 1 ml
B. Examine the chromatograms obtained in the test for                       of acetone R.
   chromatographic profile.                                                 Column :
   Results : the characteristic peaks in the chromatogram                   — material : fused silica,
   obtained with the test solution are similar in retention                 — size : l = 30 m (a film thickness of 1 µm may be used)
   time to those in the chromatogram obtained with the                          to 60 m (a film thickness of 0.2 µm may be used),
   reference solution.                                                          Ø = 0.25-0.53 mm,
                                                                            — stationary phase : macrogol 20 000 R.
TESTS
                                                                            Carrier gas : helium for chromatography R.
Relative density (2.2.5) : 0.850 to 0.858.                                  Flow rate : 1.0 ml/min.
Refractive index (2.2.6) : 1.473 to 1.476.                                  Split ratio : 1:100.
Optical rotation (2.2.7) : + 57° to + 70°.                                  Temperature :
                                                                                                   Time               Temperature
                                                                                                   (min)                 (°C)
                                                                             Column                 0-6                    45
                                                                                                    6 - 21             45 → 90
                                                                                                   21 - 39             90 → 180
                                                                                                   39 - 55                180
                                                                             Injection port                               220
                                                                             Detector                                     220

                                                             Detection : flame ionisation.
                                                             Injection : 0.5 µl of the reference solution and 0.2 µl of the
                                                             test solution.
                                                             Elution order : order indicated in the composition of the
                                                             reference solution. Record the retention times of these
                                                             substances.
                                                             System suitability : reference solution :
                                                             — resolution : minimum 1.5 between the peaks due to
                                                                 β-pinene and sabinene and minimum 1.5 between the
                                                                 peaks due to geranial and geranyl acetate.
                                                             Using the retention times determined from the chromatogram
                                                             obtained with the reference solution, locate the components
                                                             of the reference solution in the chromatogram obtained with
                                                             the test solution.
                                                             Determine the percentage content of these components. The
                                                             percentages are within the following ranges :
Figure 0620.-1. – Typical spectrum of lemon oil for the test — β-pinene : 7.0 per cent to 17.0 per cent,
                       for absorbance                        — sabinene : 1.0 per cent to 3.0 per cent,
Absorbance (2.2.25). Dissolve 0.250 g of the substance to    — limonene : 56.0 per cent to 78.0 per cent,
be examined in alcohol R, mix and dilute to 100.0 ml with    — γ-terpinene : 6.0 per cent to 12.0 per cent,
the same solvent. Measure the absorbance over the range      — β-caryophyllene : maximum 0.5 per cent,
260 nm to 400 nm. If a manual instrument is used, measure — neral: 0.3 per cent to 1.5 per cent,
the absorbance at 5 nm intervals from 260 nm to about
12 nm before the expected absorption maximum, then at        — α-terpineol: maximum 0.6 per cent,
3 nm intervals for 3 readings and at 1 nm intervals to about — neryl acetate : 0.2 per cent to 0.9 per cent,
5 nm beyond the maximum and finally at 10 nm intervals to — geranial: 0.5 per cent to 2.3 per cent,


1896                                                                 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 5.0                                                                                               Leucine



— geranyl acetate : 0.1 per cent to 0.8 per cent.                  Ninhydrin-positive substances. Examine by thin-layer
Residue on evaporation (2.8.9) : 1.8 per cent to 3.6 per cent      chromatography (2.2.27), using a TLC silica gel plate R.
after heating on the water-bath for 4 h.                           Test solution (a). Dissolve 0.10 g of the substance to be
                                                                   examined in 0.1 M hydrochloric acid and dilute to 10 ml
STORAGE                                                            with the same acid.
In a well-filled, airtight container, protected from light, at a   Test solution (b). Dilute 1 ml of test solution (a) to 50 ml
temperature not exceeding 25 °C.                                   with water R.
LABELLING                                                          Reference solution (a). Dissolve 10 mg of leucine CRS in
                                                                   0.1 M hydrochloric acid and dilute to 50 ml with the same
The label states, where applicable, that the contents are          acid.
Italian-type lemon oil.
                                                                   Reference solution (b). Dilute 5 ml of test solution (b) to
                                                                   20 ml with water R.
                                                             Reference solution (c). Dissolve 10 mg of leucine CRS and
                                                01/2005:0771 10 mg of valine CRS in 0.1 M hydrochloric acid and dilute
                                                             to 25 ml with the same acid.
                        LEUCINE                                  Apply separately to the plate 5 µl of each solution. Allow
                                                                 the plate to dry in air. Develop over a path of 15 cm using a
                                                                 mixture of 20 volumes of glacial acetic acid R, 20 volumes
                         Leucinum                                of water R and 60 volumes of butanol R. Allow the plate
                                                                 to dry in air, spray with ninhydrin solution R and heat at
                                                                 100 °C to 105 °C for 15 min. Any spot in the chromatogram
                                                                 obtained with test solution (a), apart from the principal spot,
                                                                 is not more intense than the spot in the chromatogram
                                                                 obtained with reference solution (b) (0.5 per cent). The test is
C6H13NO2                                                Mr 131.2 not valid unless the chromatogram obtained with reference
                                                                 solution (c) shows two clearly separated spots.
DEFINITION                                                       Chlorides (2.4.4). Dissolve 0.25 g in water R and dilute to
Leucine contains not less than 98.5 per cent and                 15 ml with the same solvent. The solution complies with the
not more than the equivalent of 101.0 per cent of                limit test for chlorides (200 ppm).
(S)-2-amino-4-methylpentanoic acid, calculated with              Sulphates (2.4.13). Dissolve 0.5 g in 3 ml of dilute
reference to the dried substance.                                hydrochloric acid R and dilute to 15 ml with distilled
                                                                 water R. The solution complies with the limit test for
CHARACTERS                                                       sulphates (300 ppm).
White or almost white crystalline powder or shiny flakes,        Ammonium (2.4.1). 50 mg complies with limit test B for
sparingly soluble in water, practically insoluble in alcohol.    ammonium (200 ppm). Prepare the standard using 0.1 ml of
It dissolves in dilute mineral acids and in dilute solutions of ammonium standard solution (100 ppm NH ) R.
alkali hydroxides.                                                                                              4

                                                                 Iron (2.4.9). In a separating funnel, dissolve 1.0 g in 10 ml
IDENTIFICATION                                                   of dilute hydrochloric acid R. Shake with three quantities,
                                                                 each of 10 ml, of methyl isobutyl ketone R1, shaking for
First identification : A, C.
                                                                 3 min each time. To the combined organic layers add 10 ml
Second identification : A, B, D.                                 of water R and shake for 3 min. The aqueous layer complies
A. It complies with the test for specific optical rotation (see with the limit test for iron (10 ppm).
    Tests).                                                      Heavy metals (2.4.8). 2.0 g complies with limit test D for
B. Dissolve 0.50 g in water R and dilute to 25 ml with the       heavy metals (10 ppm). Prepare the standard using 2 ml of
    same solvent. The solution is laevorotatory.                 lead standard solution (10 ppm Pb) R.
C. Examine by infrared absorption spectrophotometry              Loss on drying (2.2.32). Not more than 0.5 per cent,
    (2.2.24), comparing with the spectrum obtained with          determined on 1.000 g by drying in an oven at 100 °C to
    leucine CRS. Examine the substances prepared as discs. 105 °C.
D. Examine the chromatograms obtained in the test for            Sulphated ash (2.4.14). Not more than 0.1 per cent,
    ninhydrin-positive substances. The principal spot in the     determined on 1.0 g.
    chromatogram obtained with test solution (b) is similar
    in position, colour and size to the principal spot in the    ASSAY
    chromatogram obtained with reference solution (a).
                                                                 Dissolve 0.100 g in 3 ml of anhydrous formic acid R.
TESTS                                                            Add 30 ml of anhydrous acetic acid R. Titrate with 0.1 M
                                                                 perchloric acid using 0.1 ml of naphtholbenzein solution R
Appearance of solution. Dissolve 0.5 g in 1 M hydrochloric as indicator, until the colour changes from brownish-yellow
acid and dilute to 10 ml with the same acid. The solution is to green.
clear (2.2.1) and not more intensely coloured than reference
solution BY6 (2.2.2, Method II).                                 1 ml of 0.1 M perchloric acid is equivalent to 13.12 mg of
                                                                 C6H13NO2.
Specific optical rotation (2.2.7). Dissolve 1.00 g in
hydrochloric acid R1 and dilute to 25.0 ml with the same
acid. The specific optical rotation is + 14.5 to + 16.5,         STORAGE
calculated with reference to the dried substance.                Store protected from light.


General Notices (1) apply to all monographs and other texts                                                                  1897

				
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