Micro-identification of the Opium Alkaloids

Sections

Physical Methods
Biological Methods
Chemical Methods
Sensitivities
PRACTICAL PROCEDURE
Discussion
Summary
Acknowledgements
APPENDIX A

Details

Author: E. G. C. Clarke, Margaret Williams
Pages: 33 to 42
Creation Date: 1955/01/01

Micro-identification of the Opium Alkaloids

E. G. C. Clarke
Margaret Williams
Chemistry Division, Department of Physiology, Royal Veterinary College, London

It is probable that more tests have been described for the opium alkaloids than for any other similar group or organic substances. Their widespread use in medicine, their toxicity, and, above all, their habit-forming properties make their detection in minute quantities a matter of more than academic interest, while the legal significance of the presence of one of these substances makes their absolute identification more important than their exact quantitative estimation. It is the purpose of this paper to review briefly the methods available for the detection of the opium alkaloids when present in microgramme quantities, and to describe the application to these bases of a microtechnique recently elaborated by the authors (Clarke and Williams[1] ).

The methods available for the micro-identification of alkaloids may be classified as physical, biological and chemical.

Physical Methods

Physical methods, which have recently been reviewed by Farmilo and Levi [2] , although often requiring expensive apparatus, have the distinct advantage that in most cases they do not destroy the material used; this leaves it available for further examination. Consequently, one can employ the entire available sample in a single test, without the subdivision necessary when chemical or biological methods are used. This is not necessarily true, however, for melting-point determination, as some alkaloids are readily decomposed by heat. In any case this method is not really delicate enough for use on the micro-scale, although Fischer and Karawia [3] and Kofler and Muller [4] describe methods using some 50 &mug of material.

Numerous methods for the identification and estimation of alkaloids have been based on the optical properties of solutions. Thus, Cahen and Feuer [5] found that they were able to estimate quantities of morphine as small as 20 &mug by electrophotometric estimation of the colour produced in a modified Deniges' reaction, while Gettler and Sunshine [6] have described a method for the colorimetric determination of alkaloids in microgramme quantities by methyl orange.

The use of spectrophotometric methods in the ultra-violet region has recently been reviewed by Farmilo [7] . There is some difference of opinion as to the sensitivity of the method. Biggs, for example, was able to make rough quantitative estimations of 1 mg quantities of morphine recovered from a Stas-Otto extract, while, replacing the Stas-Otto process by a special extraction technique, Berman and Wright [9] Were able determine as little as 1 &mug of certain alkaloids. Their method unfortunately fails altogether with some substances, notably morphine and heroin. Identification of alkaloids by means of their infra-red spectra does not appear to have been applied on the microgramme scale (Marion, Ramsay and Jones [10] ; Pleas, Harley and Wibberley [11] .

Little use has been made of fluorescence as a means of identification, although Chase and Pratt [12] have applied it to powdered vegetable drugs, while Hansen [13] states that1 &mug of morphine on filter paper may be recognized by the blue zone it gives in ultra-violet light.

The properties of crystals also provide useful means of identification. Keenan [14] , for example, has recorded the optical properties of the crystals of certain derivatives of a number of important alkaloids. Powdered crystals may also be used for the production of X-ray diffraction patterns. This method has high sensitivity; Barnes [15] used 10-&mug samples while Gross and Oberst [16] obtained positive results with morphine using less than 1 &mug of material.

Paper chromatography affords a further means for the isolation and identification of alkaloids, based on the variation in R f value. By this method 20 &mug or less may be detected (Munier and Macheb&oeliguf [17] ; Curry and Powell [18] ). Borke and Kirch [19] record a similar sensitivity for surface chromatography.

Biological Methods

The biological detection of morphine depends on the "Mauseschwanzphänomen" first recorded by Straub [20] .

He noticed that mice which had received a subcutaneous injection of morphine exhibited certain definite signs, the most characteristic being the catatonic rigidity of the tail, which is carried in an S-shaped curve. This phenomenon was investigated by Herrmann [21] , who showed that the effect was roughly quantitative, the smallest quantity of morphine capable of detection being 5 &mug. Other opium alkaloids produced similar effects, although to a widely varying extent.

Maier [22] found that the duration of the " tail reaction " gave an approximate measure of the dose of morphine administered, but that a more accurate estimation could be made by finding the threshold of reaction. By this means, 20 &mug of morphine could be estimated quantitatively. Keil and Kluge [23] , interpreting their results graphically, found that they were able to estimate quantities of morphine as small as 12 &mug with an accuracy of 5%.

The detection of narcotics by bio-assay has been developed by Munch [24] [25] , who has used this method for the investigation of " doping " in racehorses. Using a standard technique, he found the threshold for a 20-g. mouse to be 1 &mug for heroin, 12 &mug for dilaudid, and 60 &mug for codeine and morphine. The method is, however, lacking in specificity. Although Munch [24] states that there is a qualitative difference between the effect of morphine and that due to heroin, it is not usually possible to distinguish between closely related substances. The great advantage of the method is the speed with which a result can be obtained. Body fluids such as saliva and urine can be injected directly without the tedious extraction and purification necessary for chemical and physical methods, and even with minimal doses effects become apparent in half an hour.

It is worth noting that Morgan and Gellhorn [26] , in a series of experiments designed to compare the sensitivities of biolo gical and chemical methods, found that, while the absolute threshold for certain substances (e.g., heroin and dilaudid) is lower for biological methods, in actual application the chemical method was not only more sensitive, but considerably more reliable.

Chemical Methods

The chemical identification of alkaloids may be carried out by means of colour or crystal tests. Both have been in use for over a century; in 1821 Orfila [27] mentioned the shape of morphine crystals, and the red colour produced with nitric acid, while in 1845 Christison [28] gave the ferric chloride and iodic acid tests for morphine, and described the shape of the crystals of the base and of various salts for morphine, codeine and narcotine. Wormley [29] in 1885 gave both colour and crystal tests in some detail. Since then, however, the colour test has become much the more popular. This may be seen by reference to various standard works on toxicology. Autenreith [30] , Glaister [31] , Kobert [32] , Nicholson [33] , Sydney Smith and Fiddes [34] , Van Oettinger [35] , and Witthaus [36] make no mention of crystal tests for the opium alkaloids. Bamford [37] , Lucas [38] , McNalley [39] , Peterson, Haines and Webster [40] and Thienes and Haley [41] give both crystal and colour tests, while the "Methods of Analysis" of the A.O.A.C.*[42] gives crystal tests only.

Both types of test have been described in almost endless variety, Bentley [43] referring to some 50 colour tests for morphine alone, while an even greater number of crystal tests for the various alkaloids of this group have been described by such authors as Vadam [44] , Stephenson [45] , Fulton [46] and Whitmore and Wood [47] . There is little to choose between colour and crystal tests from the point of sensitivity. In the case of the former, with morphine as an example, the limit of the Marquis test is variously put at 1 µg (Hansen [13] ), 2 µg (Fulton [48] ), or 5 µg (Farmilo, Levi, Oestreicher and Ross [49] ). For Froehde's test, Hansen [13] gives 0.1 µg, Wormley [29] 0.6 µg, Farmilo et al. [49] 1 µg, and Fulton [50] 0.3 µg, the last-named being for Buckingham's modification of the test. For the crystal test, Fulton [51] puts the limit at 0.1 µg for morphine with iodine reagent M-2, while Lucas [52] gives it as 1 µg or less with Marm?'s reagent.

In spite of the popularity of the colour test, and the fact that it is of equal sensitivity, in our opinion the crystal test is by far the more satisfactory. A pure substance, taken in sufficient quantity, may certainly give a series of definite colours with a certain reagent. The same substance, however, when recovered by means of a Stas-Otto extraction, is usually contaminated with material readily charred by sulphuric acid, which masks to a greater or less extent the colours due to the alkaloid. And although purification presents few difficulties when milligrammes are available, on the microgramme scale repeated manipulation tends to reduce the material to vanishing-point. Furthermore, the colour test is subjective, as the transient colour changes may be seen differently by different observers. The crystal test, on the other hand, is objective. The microcrystals are a definite entity and may be photographed for permanent record.

*Association of Official Agricultural Chemists (Ed. note).

Sensitivities

In comparing the sensitivities of these methods several points must be borne in mind. The usefulness of a test depends not only on its sensitivity, but also on its specificity and its range-i.e., the number of alkaloids for which individual results can be obtained. Some tests are more fully diagnostic than others. Thus, with the physical tests the X-ray diffraction pattern may afford complete identification, whereas the melting-point serves only to confirm a diagnosis already made. Biological tests, as already stated, are almost completely lacking in specificity and the sensitivity claimed for them is apparent rather than real, as no deductions can be drawn from the results of an experiment carried out with a single mouse. In the case of chemical tests the questions of specificity and range depend largely on the choice of reagent which is discussed in detail below.

PRACTICAL PROCEDURE

1. Crystal Test

  1. Reagents

    With crystal tests, identification depends on recognition of the characteristic shape and arrangement of the crystals formed when a suitable reagent is added to a solution of the alkaloid. The success of the test depends to a great extent on the correct choice of reagents. As stated previously, several hundred reagents have been described. In addition to those mentioned by the authors cited above, others are given by Bachmann [53] , Denoel and Soulet [54] , Duquenois and Faller [55] , Fulton and Dalton [56] , Janot and Chaigneou [57] , Levi and Farmilo [58] , Oliverio [59] , Putt [60] , Rosenthaler [61] , Uffelie [62] , Wachsmuth [63] , Wagenaar [64] and White [65] . Use of all these reagents as a general routine is obviously impracticable, as it would entail excessive subdivision of the material available. In selecting the reagents most likely to prove useful, their specificity and range must be considered. Some reagents, such as zinc chloride or sodium nitroprusside, give crystalline derivatives with but few alkaloids. That is to say, their specificity is high but their range limited. Reagents of this type are most useful for confirmatory purposes. Other reagents, such as Reinecke's salt, react with most alkaloids but give crystals so similar in appearance as to be almost useless for purposes of differentiation. Others again, such as platinum chloride and potassium bismuth iodide, react with a reasonable number of alkaloids to give crystals varying so widely in form that they provide a useful basis for identification. This class of reagent is obviously the most useful for routine testing. In practice, we find that the use of some 20 of these reagents is sufficient to identify provisionally all the more common alkaloids. Details of those most useful in the case of the alkaloids of opium are given in Appendix A.

    It will be noted that we have abandoned the use of such terms as " Wagner's reagent " and " Marm?'s reagent " as, in our opinion, the indiscriminate use of these can lead to considerable confusion.

  2. Technique

    The micro-technique recently introduced by the authors [1] is carried out as follows :* The test material is dissolved in 1% acetic acid or hydrochloric acid. A microdrop (volume approximately 0.1 cu. mm.) of this solution is transferred to a cover slip by means of a glass rod 1 mm. in diameter. A similar drop of reagent is added and the two mixed. The cover slip is inverted and placed on a cavity slide where it rests on thin glass distance pieces cemented on either side of the depression as shown in the diagram. The hanging drop is now examined under the microscope. As soon as a precipitate can be seen, the cover slip is ringed with 25% gum-arabic solution to prevent further evaporation. The drop is then examined every few hours and its appearance noted when crystallization is complete. This may take 24 hours or more. Once tentative identification has been made, similar crystals should be prepared from a known sample of the alkaloid and compared with those from the test solution. It is essential that all glassware should be well cleaned and thoroughly rinsed, as traces of synthetic detergents may interfere with precipitation (Casidio and Gallo [66] , Janot, Goutarel and Decay [67] ).

*See Fig.1.

Table 1

Alkaloid

Gold bromide

Lead iodide

Mercuric chloride

Platinum chloride

Platinum iodide

Pot. bismuth iodide

Pot. cadmium iodide

Pot. chromate

Pot. iodide

Pot. mercury iodide

Pot. tri-iodide ( 1 )

Pot. tri-iodide (2)

Pot. tri-iodide (3)

Sodium carbo-nate

Sodium nitro-prusside

Apomorphine
X
a
a
a
a
X
a
a
a
X
o
a
o
a
a
Benzyl-morphine
a
X
a
a
a
a
a
a
X
a
a
X
a
X
a
Codeine
a
X
X
a
a
a
X
-
X
X
X
-
X
-
o
Cotamine
X
X
X
X
o
a
a
-
-
X
X
o
X
-
-
Dihydro-codeine
-
-
o
-
a
X
o/a
-
-
a
o
-
o
-
-
Dihydro-codeinone
a
X
X
a
a
X
a
-
-
a
a
X
o/a
-
o
Dihydro-hydroxy-codeinone
a
-
o
X
-
a
a
-
-
a
X
-
o/a
X
-
Dihydro-morphine
a
-
o
a
X
a
o
-
-
X
o
-
o
-
-
Dihydro-morphinone
a
-
o
a
a
a(X)
a
-
-
a
o
-
o
-
X
Ethyl-morphine
a
-
X
a
a
a
X
-
-
X
-
-
X
-
-
Ethyl-narceine
a
a
o
a
o
a
o/a
X
X
a
o
X
o
o
o
Heroin
a
a
X
X
a
X
a
X
-
a
o/a
-
o/a
X
o
Morphhie
a
-
X
o
a
X
X
X
-
a(X)
X
-
o
-
-
Narceine
a
X
X
X
o
a
o
X
X
o/a
o
X
o
-
-
Narcotine
a
X
o/a
a
a
a
a
X
o
a
o/a
a
o/a
X
o
Neopine
a
-
X
-
a
a
X
-
-
X
X
-
X
-
-
Papaverine
a
o/a
X
X
a
a
X
o
X
X
a
X
a
o
o
Pseudo-morphine
a
X
X
X
X
X
a
o/a
X
a
a
X
a
-
X
Thebaine
a
o/a
o/a
X
o/a
a
a
a
o
a
a
-
a
X
o/a

X = Crystals; a = Amorphous; o = Oil droplets; o/a = Oily amorphous; a(X) = Crystals form from more dilute solutions; - = No precipitate.

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  1. Results

    Table 1 shows the results obtained by treating some of the more important opium alkaloids with fifteen different reagents, under comparable conditions. In each case a microdrop of a 1% solution of the alkaloid (containing 1 µg) was mixed with a similar drop of reagent. The slide was sealed immediately to prevent evaporation and the appearance recorded after twenty-four hours.

    Table 2 gives the appearance of the crystals and the sensitivity of the test, the latter quantity being the minimum amount of alkaloid required to give recognizable crystals. In order to obtain these figures serial dilutions of the alkaloidwere employed, sealing of the slide being deferred until the drop had been concentrated by evaporation.

    Some of the crystals obtained are shown in Plates 1-12, the weight of material used in the test being indicated.

FIGURE 1

2. Colour Tests

As already stated, in our opinion colour tests are less reliable than crystal tests, although they have considerable value for confirmatory purposes. Details of these tests are given by such authors as Bamford [38] , Fulton [50] , Jackson [68] , Pesez [69] and Taylor [70] .

The most widely used colour reactions are those in which a solution of some substance in concentrated sulphuric acid is added to a minute portion of the solid alkaloid and the resulting colour changes noted. Many substances have been suggested for this purpose, the more important including formaldehyde (Marquis [71] ), ammonium molybdate (Froehde [72] ), selenious acid (Mecke [73] , Lafon [74] ), p-dimethyl-aminobenzaldehyde (Wasicky [75] ), sodium tungstate (Reichard [76] ), titanium dioxide (Flueckiger [77] ), and sucrose (Schneider [78] , Weppen [79] ).

As we have reported previously [1] , we find that the sensitivity of most of these tests can be increased if the following procedure be adopted, Froehde's test being taken as an example. A microdrop of the test solution is placed on an opal-glass plate and a similar drop of a 0.5% aqueous solution of ammonium molybdate added. After evaporation, a microdrop of concentrated sulphuric acid is applied to the residue, and the colour changes noted. In the same way the tests ascribed to Mecke, Reichard and Schneider-Weppen may be carried out with aqueous solutions containing 0.5% of selenious acid, 1% sodium tungstate and 10% of sucrose respectively. For Wasicky's test a 10% solution of p-dimethylamino-benzaldehyde in glacial acetic acid is used.

Table 2

Reagent

Appearance of crystals

Sensitivity (µg)

Apomorphine
 
 
Gold bromide
Serrated crosses
0.0005
Potassium bismuth iodide
Rosettes of dark needles
0.005
Potassium mercury iodide
Bunches of needles
0.05
Benzylmorphine
 
 
Lead iodide
Hedgehogs (2 days)
0.25
Sodium carbonate
Rosettes of rods
0.05
Potassium tri-iodide (2)
Rosettes of plates (2 days)
0.25
Potassium iodide
Rosettes of needles or plates
0.025
Codeine
 
 
Potassium tri-iodide (1)
Feathery rosettes
0.05
Potassium cadmium iodide
Small rosettes-small plates
0.01
Potassium mercury iodide
Irregular plates
0.025
Lead iodide
Rosettes of rods (2 days)
0.5
Mereuric chloride
Irregular needles in bunches
0.25
Potassium iodide
Rosettes of needles
1.0
Potassium tri-iodide (3)
Dense rosettes
0.25
Cotarnine
 
 
Platinum chloride
Rods
0.025
Mercuric chloride
Bunches of needles
0.05
Gold bromide
Yellow rods
0.1
Potassium tri-iodide (1)
Very small plates
1.0
Potassium tri-iodide (3)
Small rods
0.005
Potassium mercury iodide
Irregular plates
0.05
Lead iodide
Needles or dendrites
0.25
Dihydrocodeine
 
 
Potassium bismuth iodide
Bunches of hexagonal plates
0.005
Dihydrocodeinone
 
 
Mercuric chloride
Dense rosettes
0.05
Potassium tri-iodide (2)
Very long rods (2 days)
0.25
Lead iodide
Dense rosettes (2 days)
0.1
Potassium bismuth iodide
Rhomboidal plates
0.01
Dihydrohydroxycodeinone
 
 
Potassium tri-iodide (1)
Small dark plates
0.0025
Sodium carbonate
Long thin needles
0.025
Platinum chloride
Rosettes of rods
0.25
Dihydromorphine
 
 
Platinum iodide
Smudge rosettes
0.025
Potassium mercury iodide
Sheaves of free needles
0.1
Dihydromorphinone
 
 
Sodium nitroprusside
Rods
0.5
Potassium bismuth iodide
Grain-like crystals (2 days)
0.05
Ethyl morphine
 
 
Mereuric chloride
Long blades
1.0
Potassium mercury iodide
Smudge rosettes
0.05
Potassium cadmium iodide
Smudge rosettes (2 days)
0.1
Potassium tri-iodide (3)
Bunches of feathery needles
0.05
Ethyl narceine
 
 
Potassium iodide
Rosettes of rods
0.025
Potassium chromate
Bunches of rods
0.1
Potassium tri-iodide (2)
Rosettes of rods
0.05
Heroin
 
 
Platinum chloride
Rosettes
0.25
Potassium bismuth iodide
Small dense rosettes (2 days)
0.005
Mercuric chloride
Fine dendrites
0.1
Potassium chromate
Smudge rosettes (hedgehogs)
0.1
Sodium carbonate
Rosettes of plates
1.0
Morphine
 
 
Potassium cadmium iodide
Sheaves of fine needles
0.01
Potassium tri-iodide (i)
Plates
0.1
Mereuric chloride
Bunches of needles
0.1
Potassium bismuth iodide
Small rods and plates
0.01
Potassium mercury iodide
Tufts of thread-like crystals
0.05
Potassium chromate
Small plates
0.5
Narceine
 
 
Platinum chloride
Rosettes of blades
0.25
Mercuric chloride
Small rosettes
0.25
Lead iodide
Thread-like crystals
0.1
Potassium chromate
Tufts of needles
0.1
Potassium tri-iodide (2)
Rosettes of needles
0.025
Potassium iodide
Fine needles
0.025
Narcotine
 
 
Sodium carbonate
Rosettes and bunches of needles
0.025
Potassium chromate
Rosettes
0.1
Lead iodide
Rosettes of fine branching needles
0.25
Neopine
 
 
Potassium cadmium iodide
Silvery rosettes - plates
0.025
Potassium mercury iodide
Oily rosettes
0.025
Potassium tri-iodide (3)
Small plates or rosettes
0.1
Potassium tri-iodide (1)
Rosettes of feathery needles
0.05
Mercuric chloride
Bunches of plates, rods and needles
0.25
Papaverine
 
 
Platinum chloride
Yellow plates (2 days)
0.05
Potassium iodide
Irregular plates
0.025
Potassium mercury iodide
Plates and squares
0.05
Potassium tri-iodide (2)
Small rosettes
0.1
Potassium cadmium iodide
Rosettes of blades
0.025
Mercuric chloride
Very small needles
0.25
Pseudomorpine
 
 
Potassium iodide
Petal-shaped crystals
0.01
Platinum iodide
Small dark rosettes (2 days)
0.25
Platinum chloride
Small rods
0.025
Lead iodide
Squares
0.025
Potassium bismuth iodide
Rhomboids and rods (2 days)
0.05
Potassium tri-iodide (2)
Plates
0.05
Mercuric chloride
Smudge rosettes - rods and plates
0.1
Sodium nitroprusside
Plates
0.1
Thebaine
 
 
Sodium carbonate
Rosettes of small plates
0.025
Platinum chloride
Dense rosettes
0.05

This modification is not practicable for Marquis' test or Flueckiger's test, which are carded out by treating the residue left by evaporating a microdrop of the test solution with a trace of the original reagent - namely, a mixture of one part of 40% formaldehyde solution with twenty parts of concentrated sulphuric acid for the former and a 0.5% solution of titanium dioxide in concentrated sulphuric acid for the latter.

Carried out in the above manner, these tests have sensitivities ranging from 1 µg to 0.025 µg, although the colour changes observed are not always the same as when larger quantities are employed. The colours obtained for 1 µg quantities of a number of alkaloids together with the absolute sensitivities are given in Table 3. It should be noted that with minimal quantities it is seldom that the whole range of colours is observed.

Examples of the crystals obtained with various reagents

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Table 3

 

Ammonium molybdate (Froehde)

Selenious acid (Mecke)

Sodium tungstate (Reichard)

Alkaloid

 

in µg

 

in µg

 

in µg

Apomorphine
Deep green - blue green
0.1
Blue black - green - brown
0.1
Black violet
0.025
Benzylmorphine
Violet - green
0.05
Green
0.1
Violet
0.25
Codeine
Blue, slowly fading
0.1
Blue green - yellow green - brown
0.5
Pale violet
0.5
Cotarnine
Pale violet - green
0.25
Yellow brown
0.5
Yellow - brown
0.5
Dihydrocodeine
Green - blue
0.1
Green - yellow brown
0.1
______
 
Dihydrocodeinone
Green - blue
0.1
Yellow green
0.25
______
 
Dihydrohydroxycodeinone
Yellow - green - blue
0.1
Orange - olive green
0.25
______
 
Dihydromorphine
Blue violet
0.025
Brown - green
0.25
Violet
0.25
Dihydromorphinone
Purple - blue - green
0.05
Orange - brown
0.25
Dark brown
0.25
Ethylmorphine
Green - blue
0.1
Green
0.5
Blue violet
0.5
Ethylnarceine
Green - blue
0.05
Green - blue
0.1
Orange - green
0.1
Heroin
Red violet - blue - light green
0.05
Blue green - olive green - brown
0.5
Deep violet
0.5
Morphine
Violet - blue - light green
0.05
Blue green - grey green
0.1
Violet
0.25
Narceine
Brown - grey - blue - green
0.05
Bright green - grey - orange
0.25
Orange - green
0.05
Narcotine
Brown - green - blue - pale green
0.05
Green - orange
0.25
Blue violet
0.25
Neopine
Blue - green
0.05
Reddish purple - brown
0.1
______
 
Papaverine
Faint green - blue
0.5
Grey - grey green fading
0.25
______
 
Pseudomorphine
Blue - violet - green
0.1
Purple - brown
0.1
Deep violet
0.1
Thebaine
Greenish brown - red brown
0.1
Green - brown - orange
0.25
Brownish green
0.25
 

Sucrose (Schneider-Weppen)

p-dimethylamino benzaldehyde (Wasicky )

Formaldehyde (Marquis)

Titanium dioxide (Flueckiger)

Alkaloid

 

in µg

 

in µg

 

in µg

 

in µg

Apomorphine
Brown
0.25
Red
0.25
Purple - black
0.05
Deep purple
0.05
Benzylmorphine
Pink
0.5
Orange
0.25
Red - purple
0.025
Purple
0.05
Codeine
Pink
0.5
Orange
0.25
Violet
0.05
_________
 
Cotarnine
Orange
0.5
_________
 
_________
 
Yellow - brown
0.5
Dihydrocodeine
Pink
0.5
Orange
0.25
Purple
0.1
_________
 
Dihydrocodeinone
Pink
1.0
Orange
0.5
Yellow - brown - purple
0.25
_________
 
Dihydrohydroxy-codeinone
Pink
1.0
Faint orange
0.1
Yellow - brown - purple
0.5
_________
 
Dihydromorphine
Pink
0.25
Bright orange
0.25
Red purple
0.025
Deep purple
0.025
Dihydromorphinone
Pink
1.0
Orange
0.25
Yellow - red - purple
0.25
Deep purple
0.025
Ethylmorphine
Pink
1.0
Bright orange
0.1
Yellow - purple - black
0.1
_________
 
Ethylnarceine
Yellow
0.5
Yellow
0.5
Brown - green - blue
0.1
Orange - brown
0.1
Heroin
Pink
0.5
Orange
0.1
Violet
0.05
Deep purple
0.025
Morphine
Pink
0.25
Orange
0.1
Violet
0.05
Deep purple
0.025
Narceine
Yellow
0.25
Yellow
0.25
Brown - deep brown - green
0.05
Bright orange - brown
0.1
Narcotine
Brown
0.1
Yellow
0.25
Bluish violet fading
0.1
Faint brown - purple
0.5
Neopine
Pink
0.25
Orange
0.1
Blue violet
0.1
Faint purple
0.5
Papaverine
Brown
0.1
_________
 
_________
 
Faint purple
1.0
Pseudomorphine
Green
1.0
_________
 
Rose red
0.1
Deep purple
0.1
Thebaine
Brown
0.025
Brown
0.25
Red - orange
0.05
Green - brown - black
0.1

Discussion

The "smallest detectable quantity" of any substance depends on a number of factors including the efficiency of the extraction process employed, the number of different tests that must be carried out and the sensitivities of these tests. Leaving the first of these factors out of consideration and assuming the second to have been reduced to a minimum by correct choice of reagents, it is obvious that any technique that increases the sensitivity of the test causes a corresponding decrease in the amount that can be identified. As the results tabled above show at least one crystal test with a sensitivity of 0.05 µg for each alkaloid mentioned, it follows that, under favourable circumstances, one should be able to identify quantities of the order of a microgramme of any of these substances, provided that they are in a reasonable state of purity.

Although most of these alkaloids may be identified without difficulty, it is not easy to differentiate between codeine and neopine. This might be expected from their structures, which differ only in the position of a double bond. We find that potassium mercury iodide is the most satisfactory reagent to distinguish between these substances. In both cases gelatinous rosettes are formed, but whereas with codeine these quickly disintegrate into small plates, in the case of neopine no such change takes place.

Summary

  1. The literature dealing with the subject is briefly reviewed.

  2. Results are given of the application to a number of the opium alkaloids of new microtechniques for colour and crystal tests.

Acknowledgements

We wish to express our thanks to Professor E. C. Amoroso for his help and encouragement and to Mr. R. F. S. Creed and Mr. H. Burgess for taking the photographs. We acknowledge gratefully gifts of alkaloids from Messrs. T. and H. Smith Ltd., and Messrs. J. F. MacFarlan and Co. Ltd. We are also much indebted to Miss Ann Stanley for technical assistance.

APPENDIX A

Reagents for microcrystalline tests

(Unless otherwise indicated, the quantities shown are dissolved in 100 ml of water)

  1. Gold bromide: 5 g gold chloride + 5 g sodium bromide.

  2. Lead iodide: Dissolve 30 g lead acetate in 100 ml of water, adjust to pH 6 with acetic acid, and saturate with lead iodide.

  3. Mercuric chloride: 5 g.

  4. Platinum chloride: 5 g.

  5. Platinum iodide: 5 g platinum chloride + 25 g sodium iodide.

  6. Potassium bismuth iodide: 5 g bismuth subnitrate + 25 g

potassium iodide in 100 ml of 2% sulphuric acid.

  1. Potassium cadmium iodide: 1 g cadmium iodide + 2 g potassium iodide.

  2. Potassium chromate : 5 g.

  3. Potassium iodide: 5 g.

  4. Potassium mercury iodide: 1.5 g mercuric iodide + 5 g potas-sium iodide.

  5. Potassium tri-iodide (1) : 2 g iodide + 4 g potassium iodide.

  6. Potassium tri-iodide (2) : 0.1 g iodine + 0.2 g potassium iodide.

  7. Potassium tri-iodide (3) : 1 g iodine + 50 g potassium iodide.

  8. Sodium carbonate: 5 g.

  9. Sodium nitroprusside: 1 g.

REFERENCES

001

CLARKE, E. G. C., and WILLIAMS, M., J. Pharm. Lond. VII, 255,1955.

002

FARMILO, C. G., and LEVI, L., Bull. Narcotics V (4), 20, 1953.

003

FISCHER, R., and KARAWIA, M. S., Mikrochem. Acta, 366, 1953.

004

KOFLER, L., and MÜLLER, F. A., Mikrochemie, 22, 43, 1937.

005

CAHEN, R., and FEUER, H., C. R. Acad. Sci., 208, 1907, 1939.

006

GETTLER, A. O., and SUNSHINE, I., Anal. Chem., 23, 779, 1951.

007

FARMILO, C. G., Bull. Narcotics, VI (3-4), 18, 1954.

008

BIGGS, A. I., J. Pharm. Lond. IV, 547, 1952.

009

BERMAN, E., and WRIGHT, H. N., A.M. A. Arch. induustr. Hyg., 8, 518, 1953.

010

MATRON, L., RAMSAY, D. A., and JONES, R.. N., J. Amer. Chem. Soc., 73, 305, 1951.

011

PLEAT, G. B., HARLEY, J. H., and WIBBERLY, S. E., J. Amer. pharm. Ass. Sci. Ed., 40, 107, 1951.

012

CHASE, C. R., and PRATT, R., J. Amer. pharm. Ass. Sci. Ed., 38, 324, 1949.

013

HANSON, A., Svensk. kem. Tidskr., 58, 10, 1946.

014

KEENAN G. L., Chem. Anal., 39, 33, 52, 79,1950; ibid., 40, 4, 28,1951.

015

BARNES, W. H., Bull. Narcotics, VI (1), 20 (2), 27, 1954.

016

GROSS, S. T., and OBERST, F. W., J. Lab. din. Med., 32, 94, 1947.

017

MUNIER, R., and MACHEBCEUF, M., Bull. Soc. Chim. biol., 31, 1144, 1949.

018

CURRY, A. S., and POWELL, H., Nature, Lond., 173, 1143, 1954.

019

BORKE, M. L., and KIRCH, E. R ., J. Amer. pharm. Ass. Sci. Ed. 42, 627, 1953.

020

STRAUB, W., Dtsch. med. Wschr., 37, 1462, 1911.

021

HERMAN, O., Biochem. Z., 39, 216, 1912.

022

MAIER L., Arch. exp. Path. Pharmak., 161, 163, 1931.

023

KEIL, W., and KLUGE, A., Arch. exp. Path. Pharmak., 174, 493,1934.

024

MUNCH, J. C., J. Amer. pharm. Ass. Sci. Ed. XXIII, 766, 1185, 1934; ibid., XXIV, 557, 1935.

025

MUNICH, J. C., SLOANE, A. B., and LATVEN, A. R., Bull. Narcotics, IV (3), 23, 1952.

026

MORGAN, C. E., and GELLHORN, A., lndustr. Engng. Chem. (Anal.), 19, 806, 1947.

027

ORFILA, M.P., A General System of Toxicology, Cox, Lond., 1821.

028

CHRISTION, R., A Treatise on Poisons, Black, Edinburgh, 1845.

029

WORMLEY, T. G., Microchemistry of Poisons, Lippincott, Philadelphia, 1885.

030

AUTENREITH, W., Detection of Poisons, Churchill, Lond., 1928.

031

GLASTER, J., Medical Jurisprudence and Toxicology, Livingstone, Edinburgh, 1950.

032

KOBERT, R., Lehrbuch der Intoxikation, Ferdinand Enke, Stuttgart, 1902.

033

NICHOLSON, J. A., Lander's Veterinary Toxicology, Ballière, Tindall & Cox, Lond., 1945.

034

SYDNEY SMITH, and FIDDES, F. S., Forensic Medicine, Churchill, Lond., 1949.

035

VON OETTINGEN, W. F., Poisoning, Heinemann, Lond, 1952.

036

WITTHAUS, R. A., Manual of Toxicology, Wood, New York, 1911.

037

BAMFORD, F., Poisons, Their Isolation and Identification. Churchill, Lond., 1951.

038

LUCAS, A., Forensic Chemistry, Arnold, Lond., 1945.

039

MCNALLY, W. D., Medical Jurisprudence and Toxicology, Saunders, Lond., 1939.

040

PETERSON, F., HAINES, W. S., and WEBSTER, R,. W., Legal Medicine and Toxicology, Vol. II, Sau nders, Lond., 1923.

041

THIENES, C. H., and HALEY, T. J., Clinical Toxicology, Kimpton, Lond., 1948.

042

Official Methods of Analysis, Ass. Off. Agric. Chem. Wash. Washington, D. C., 1950.

043

BENTLEY, K. W., The Chemistry of the Morphine Alkaloids, Clarendon, Oxford, 1954.

044

VADAM, M., J. Pharm. Chim., 4, 485, 1896; ibid., 5, 100, 1897.

045

STEPHENSON, C. H., Some Micro-chemical Tests for Alkaloids, Griffin, Lond., 1921.

046

FULTON, C. C., Amer. J. Pharm., 104, 244, 1932; ibid., 112, 51, 134, 1940.

047

WHITMORE, W. F., and WOOD, C. A., Mikrochemie, 27, 249, 1939.

048

FULTON, C. C., Amer. J. Pharm., 109, 219, 1937.

049

FARMILO, C. G., LEVI, L., OESTREICHER, P. M. L., and Ross, K. J., Bull. Narcotics IV (4), 16, 1952.

050

FULTON, C. C., J. Lab. clin. Med., 23, 625, 1938.

051

FULTON, C. C., J. Lab. clin. Med., 23, 622, 1938.

052

LUCAS, G. K. W., Canad. J. Res., 28, B, 37, 1950.

053

BACHMANN, G., Pharm. Ztg., 84, 305, 1948.

054

DENOEL, A., and SOULET, U., J. Pharm. Belg., 1, 34, 50, 66, 1942.

055

DUQUENOIS, P., and FALLER, Mlle, Bull. Soc. chim. Fr., 6, 998, 1939.

056

FULTON, C. C., and DALTON, J. B., J. of Crim. Law and Criminology, 32, 358, 1941.

057

JANOT, M., and CHAIGNEOU, M., C. R. Acad. Sci., 229, 69, 1949.

058

LEVI, L., and FARMILO, C. G., Canadian J. of Chem., 30, 783, 1952.

059

OLIVERIO, A., Ann., Chim., appl., 28, 353, 1938.

060

PUTT, E. B., J. industr. Engng. Chem., 4, 508, 1912.

061

ROSENTHALER, L., Arch. Pharm. CCLXV, 319, 1927.

062

UFFELIE, O. F., Chem. Weekbl., 41, 101, 1945.

063

WACHSMUTH, H., J Pharm. Belg., 8, 283, 1953.

064

WAGENAAR, G. H., Pharm. Weekbl., 76, 276, 1939.

065

WHITE, E. P., Industr. Engng. Chem. (Anal.), 13, 509, 1941.

066

CASADIO, S., and GALLO, U., Boll. chim.-farm., 91, 177, 1952.

067

JANOT, M. M., GOUTAREL, R., and DECAY, A ., Ann. pharm, franç., 5, 267, 1947.

068

JACKSON, K. E., Industr. Engng. Chem. (anal.), 10, 380, 1938.

069

PESEZ, M., Ann. Chim. anal., 24, 153, 1942.

070

TAYLOR, F. O., Allen's Commercial Organic Analysis, VII, 665, Churchill, Lond., 1929.

071

MARQUIS, Z. anal. Chem., 38, 467, 1899.

072

FROEHDE, A., Arch. Pharm., 176, 54, 1866.

073

MECKE, Z. Off. Chem., 5, 351, 1899, cited in Merck's Index, Merck & Co., Inc. Rahway, N.J., 5 th Ed., 1940. Original reference not available.

074

LAFON, C. R. Acad. Sci. C., 1543, 1886.

075

WASICKY, Z. anal. Chem., 54, 394, 1915.

076

REICHARD, Z.anal. Chem., 42, 95, 1903.

077

FLUECKIGER, F. A., Feb. 1880, cited in the Dispensatory of the U.S.A., edited by Remington et al. , Lippincott, Philadelphia, 20 th Ed., 1918.

078

SCHNEIDER, R., J. prakt. Chem. (2) 6, 455, 1873.

079

WEPPEN, Arch. Pharm., 205, 112, 1874.