Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 71 and End)

8.68.6
© Springer-Verlag Berlin Heidelberg 2005
II.8.6 Methemoglobin
by Keizo Sato
Introduction
Determination of methemoglobin ( Met-Hb) in blood is important for the diagnosis of poison-
ing by oxidants, such as nitrite, nitrate, chlorate, chlorite, alkyl nitrites, nitroglycerin, aniline
and other compounds. In 1938, Evelyn and Malloy [1] had devised a photoelectric method for
determination of Met-Hb in blood. Minor modi cations of this method were made by several
researchers to increase sensitivity [2–4].  ese methods are based on a phenomenon that the
absorbance maximum of weakly acidic Met-Hb at 630 nm disappears by addition of cyanide.
Met-Hb concentration can be easily determined as the ratios of the absorbance changes in-
duced by cyanide before and a er addition of potassium ferricyanide.
In forensic science practice, there are many cases, in which Met-Hb has to be measured
for blood specimens containing high concentrations of carboxyhemoglobin (CO-Hb); e.g.,
especially in the cases of  re and exhaust gas poisoning [5]. However, the above methods
[1–4] are not suitable for specimens containing high levels of CO-Hb. In 1981, the author
et al. [6] developed a modi cation of the methods, which enabled accurate determination
of blood Met-Hb even in the presence of high concentrations of CO-Hb by using about a
100-fold excess of potassium ferricynade to be added to the Hb iron. In this chapter, a simple
procedure of the modi ed spectrophotometric method for Met-Hb is brie y described.
Reagents and their preparation
• Potassium ferricyanide, 4 % (w/v) in distilled water. It should be stored in a dark bottle, and
be prepared monthly.
• Phosphate bu er solution, 0.1 M, pH 6.8.
• Phosphate bu er solution, 40 mM, pH 6.9. It can be prepared by dilution of two parts of
the above reagent with three parts of distilled water.
• Potassium cyanide, 5 % (w/v) dissolved in the above 40 mM phosphate bu er solution. It
should be prepared just before use.
Analytical instrument
A Hitachi 557 dual-wavelength spectrophotometer

a
(Hitachi, Ltd., Tokyo, Japan).
Procedure
i. A 6-mL volume of distilled water is placed in a test tube. A 0.2-mL volume of whole blood
is added to the tube and mixed well.
656 Methemoglobin
ii. A er allowing the tube to stand for 5 min, 4-mL of 0.1 M phosphate bu er solution is
added to the mixture and mixed well.
iii.  e hemolysate is centrifuged at 3,000 rpm for 10 min, and the clear supernatant is trans-
ferred to another test tube.  e pH of the supernatant should be around 6.9.
iv. Four 4-mL volume cuvettes of the same type are cleaned well by washing with distilled
water.  e four cuvettes are designated as A, B, C and D.
v. To cuvettes A and B, a 0.5-mL volume each of distilled water is added. To cuvettes C and
D, a 0.5-mL volume each of 4 % potassium ferricyanide solution is added.
vi. To cuvettes A and C, a 3-mL volume each of 40 mM phosphate bu er solution is added.
To cuvettes B and D, a 3-mL volume each of the supernatant of the above hemolysate is
added. Each cuvette is mixed well.
vii.  e absorbance of cuvette B at 630 nm is read using cuvette A as reference, and this read-
ing is A
1
. A er allowing cuvette D to stand for 10 min, the absorbance at 630 nm is read
using cuvette C as reference, and this reading is A
3
.
viii. To all cuvettes, a 30-µL volume each of 5 % potassium cyanide solution is added and
mixed well.
ix. A er allowing the cuvettes to stand for 2 min, the absorbances of cuvettes B and D at
630 nm are read using cuvettes A and C as references, respectively.  ese readings are A
2

and A
4
, respectively.
x.
 e percentage of Met-Hb is calculated by the following equation: Met-Hb % = 100 (A
1
–A
2
)/
(A
3
–A
4
).
Assessment and some comments on the method
 e present method for analysis of Met-Hb in blood is simple and rapid, and is not interfered
with by the coexistence of high concentrations of CO-Hb [6]. As low as about 0.2 % of Met-Hb
can be measured accurately.
However, when high concentrations of sul emoglobin (SHb) may interfere with the pres-
ent assay [7]; but SHb concentrations in putre ed blood not older than 7 days are usually not
high, and do not probably in uence the present assay. Caution should be, therefore, made for
blood specimens obtained from living subjects or cadavers of sul de or polysul de poisoning
(see the chapter “Hydrogen sul de and its metabolite” of this book).
Met-Hb in blood is not stable at room temperature and even at –30 °C.
When analysis of Met-Hb has to be made later, the blood specimens should be stored at
–80 °C or even lower temperature [8], or at –30 °C in the presence of a cryoprotective solution
b

[9, 10].
Toxic and fatal concentrations

Clinical symptoms according to the concentrations of Met-Hb are presented in >Table 5.1 of
the previous chapter (Chapter II.8.5, “Nitrate and nitrite compounds”).
657Methemoglobin
Notes
a) Any type of spectrophotometers can be used, regardless of manufacturers.
b)  e cryoprotectant is aqueous solution containing 28 % glycerol, 3 % mannitol and 0.65 %
NaCl. It should be added to blood samples at the ratio of 1:1.
References
1) Evelyn KA, Malloy HAT (1938) Microdetermination of oxyhemoglobin, methemoglobin and sulfhemoglobin in
a single sample of blood. J Biol Chem 126:655–662
2) Leahy T, Smith R (1960) Notes on methemoglobin determination. Clin Chem 6:148–152
3) van Kampen EJ, Zijlstra WG (1965) Determination of hemoglobin and its derivatives. Adv Clin Chem 8:141–
187
4) Hegesch E, Gruener S, Cohen R et al. (1970) A sensitive micromethod for the determination of methemoglobin
in blood. Clin Chim Acta 30:679–682
5) Katsumata Y, Aoki M, Oya M et al. (1980) Simultaneous determination of carboxyhemoglobin and methemoglo-
bin in victims of carbon monoxide poisoning. J Forensic Sci 25:546–549
6) Sato K, Katsumata Y, Aoki M et al. (1981) A practical method for the accurate determination of methemoglobin
in blood containing carboxyhemoglobin. Forensic Sci Int 17:177–184
7) Sato K (1983) A method for determination of methemoglobin in blood containing carboxyhemoglobin. Jpn
J Legal Med 37:133–144 (in Japanese with an English abstract)
8) Sato K, Tamaki H, Okajima H et al. (1988) Long-term storage of blood samples as whole blood at extremely low
temperatures for methemoglobin determination. Forensic Sci Int 37:99–104
9) Sato K, Katsumata Y, Aoki M et al. (1987) Long-term storage of blood samples at freezing temperatures in the
presence of a cryoprotectant for hemiglobin assay. Biochem Med Metab Biol 37:190–196
10) Sato K, Tamaki K, Tsutsumi H et al. (1990) Storage of blood for methemoglobin determination: comparison
of storage with a cryoprotectant at –30
129–134
°C and without any additions at –80 °C or –196 °C. Forensic Sci Int 45:
659Subject index

© Springer-Verlag Berlin Heidelberg 2005
Subject index
A
α-bromoisovalerylurea 293
5α-cholestane 188–190
α-DEX 120 150
α-hydroxytriazolam 283,
287, 289, 290
α-methylene-γ-butyrolac-
tone 248
acebutolol 373
acephate 537
2-acetamidophenol 339,
574
acetaminophen 335, 336,
338, 393
acetonitrile extraction 538
acetylacebutolol 373
acetylcholinesterase 84, 535,
559, 619
acetylcholine receptors 365
6-acetylmorphine 195, 198,
201
6-acetylmorphine-TMS 201
acetylsalicylic acid 343, 344,
346, 348, 392
acid 187, 377
acidosis 123
aconine 458, 461
aconite toxins 455

aconitine 455, 458, 459
aconitum alkaloids 455
acylation 30
adhesives 149
adulterant 491
advanced automated sample
processors 31
AED 81
A-esterase 535
air bags 629
alachlor 529
alcohol 135
alcohol dehydrogenase 123
ALD-type 2 137
aldehyde dehydrogenase
123, 137
aldicarb 566
aldrin 529
alkylated adducts 85
alkylated DNAs 85
alkylation 30
alkyl nitrites 153, 655
allobarbital 305, 308
allylesterase 624
allyxycarb 566
alminoprofen 325, 330, 332
alprazolam 284, 285, 290
alternative specimens 9
Amanita neoovoidea 480
Amanita pantherina 469

Amanita phalloides 469
Amanita verna 469, 474
Amanita virosa 469, 473
amanitin 473, 477, 479
amido-type local anesthetic
385
1-amino-4- phenylbutane
173
2-amino-6-hydroxymethyl-
8-hydroxyquinazoline
482
aminomethyl phosphonic
acid 546
7-aminonitrazepam 19
5-aminoquinoline 499
amitriptyline 271, 274, 276
amniotic  uid 12
amobarbital 301, 303, 305,
308, 309
amoxapine 271, 274, 276
amoxicillin 398
amphetamine 171, 172, 178
ampicillin 395
amyl nitrite 153
analgesic-antipyretics 325
analysis mailing list 52
anaphylactic shock 395
angel’s trumpet 516
angel dust 241
angina pectoris 153

anhydride 208
aniline 151, 655
anti-in ammatory 325
antidepressants 271, 272,
274, 276
antiepileptics 351, 355
antimony 644
antitussives 195
9-anthracenecarboxylic acid
331, 332
an Oasis HLB 3 cc 260
AP 171, 179–182
APAP 335
APAP-3-mercapturic acid
337, 338
APAP-glucuronide 335, 336,
338
APAP-N-sulfate 335, 336,
338
APC 564
APCI 40, 525
apronalide 295, 421
ArCl
+
42
ArCl
+
ion 645
argride 42
arrythmia 458

arsenic 637
arsenic (III) oxide 637
arsenic poisoning 645
arsenious acid 637, 646
arsenite 637, 646
arsenocholine 646
arylesterase 535
As
2
O
3
637
ASA 343
Asahi-Pak GS-320 366
ascorbic acid 117
aspirin 343
660 Subject index
atenolol 370, 373
atmospheric chemical
ionization 40
atmospheric pressure
chemical ionization 364,
525, 541
atomic absorption
spectrometry 43
atrazine 597
atropa belladonna 516
atropine 509, 514
atropine-d
3

509
automated pretreatments
31
autosuppressor 629
azeotropic e ects 501
B
β-adrenergic receptor
antagonists 369
β-blockers 369, 371, 373,
374, 375
β
1
-blockers 369
β
2
-blockers 369
β-CD 173
β-cyclodextrin 173, 582
β-DEX 225 174
β-glucuronidase 184, 231,
238, 232, 287, 479, 581
β-glucuronidase K-2 372
β-glucuronidase-sulfatase
337
β-hydroxyethyltheophylline
395
β-lactam antibiotics 395
β-phenylethylamine 18
bacteria 18
bacterial food poisoning 2

bacterial translocation 18
BAL 85
barbital 303, 308
barbiturates 301, 302,
304–307, 309
BASTA Fluid
®
545
1,4-BD 247, 250
BE 207, 211, 214, 216
belladonna alkaloids 509
benactyzine 315
bensil 5C
18
-C 426
benthiocarb 564
bentone-34 150
benzalkonium chlorides
407, 410
benzene 149, 151, 165
benzodiazepines 283, 285
benzophenones 287
benzoylaconine 458, 460
benzoylecgonine 207
benzoylhypaconine 460
benzphetamine 171
benzyldimethyldodecylam-
monium 408
benzyldimethylhexadecyl-
ammonium 408

benzyldimethyltetradecyl-
ammonium 408
benzylpenicillin 395
betaxolol 373
bialaphos 545
bismuth 644
bisoprolol 373, 375
bis(1-phenyl-3-methyl-5-
pyrazolone) 114
bleaching powder 403
blood 2
blood ethanol 138
body builders 247
Bond Elut C
1
366
Bond Elut C
18
593
Bond Elut CBA 363
Bond Elut Certify 197
Bond Elut Certify
®
II 191,
556
Bond Elut Certify LRC 479
Bond Elut Glass 242
Bond Elut SCX 233, 486
Bond Elut SI 331, 457
bone marrow 297

borax 431
boric acid 431
boric ball 431
brain-to-blood ratios 213
breath ethanol 137, 139, 141
bromadiolone 599, 600
bromazepam 284, 285
bromisovalum 293, 295,
296, 421
Bromocresol green reagent
325
2-bromohexanoylurea 295,
297
2-bromoisobutyrylurea 298
bromovalerylurea 293
bromperidol 263, 265, 268
brompheniramine 320
brotizolam 284, 285
Brovarin 293
BSA 198, 201
BSTFA 75, 191, 205
bupivacaine 384, 387
bupranolol 373
1,4-butanediol 247
butyl nitrite 153
butyl rubber septa 167
butyrophenones 263
by-products 83
BZP 171
C

C
9
base 482, 484, 489
Ca(ClO)
2
403
cadmium 637
ca eine 229, 491, 494, 495
calcium hypochlorite 403
calcium oxalate 452
camphor 443
cannabichromene 189
cannabidiol 187
cannabinoids 187
cannabinol 187
cannabis 187
Cannabis sativa L. 187
Capcell Pak C
18
MG 293
Capcell Pak C
18
UG-80 259
Capcell Pak SCX 231
capillary electrophoresis 60,
177, 181, 403
capillary HPLC 38
661Subject index
carbamate pesticides 559
carbamazepine 271, 272,

274, 276, 278, 351, 353,
354
carbamazepine-diol 353,
354
carbamazepine-epoxide 353,
354
carbaryl 529
carbendazole 563
carbinoxamine 315, 384
carbofuran 567
carbon monoxide 91, 144
carboxyesterase 535
carboxyhemoglobin 91, 144,
655
carboxylic acid 30
cardiac glucosides 519
cardiac puncture 5
cardiopulmonary resuscita-
tion 22
carry-over 533
carteolol 373, 375
catechol 3-hydroxyamino-
phen 337, 338
CBC 189
CBD 187, 189
CBN 187, 189
CBP-1 311, 331
CE 172, 177, 403
CE/MS 514, 515
cefotaxime 398

cefoxitin 398, 400
cefuroxime 398, 400
celiprolol 373
cephalexin 398, 400
cephaloridine 398, 400
cephalosporins 395
cephamycins 395
cephems 395
cerebrospinal  uid 479
changed form of MEGX
387
chasmanine 455
ChemElut 500, 507
chemical ionization 40
chemical warfare agents 69,
609
chemical weapons 69, 73,
609
Chemical Weapons
Convention 69, 609
chiral analysis 181, 514
Chiral Drug Column 177
chiral GC/MS 174, 182
chiral LC/MS 177, 180
chloramine T 113
chlorate 655
chlorate ion 404
chlordene 529
chlordene-cis 530
chlordene-trans 530

chlordiazepoxide 285
chlorine gas 403
chlorite 655
chlorocyanogen 115
chloroephedrine 171
chlorofenavinphos 537
chloro uorocarbon 143
chloroform 143, 145
chloromethylephedrine 171
1.8-chlorotheophylline 391
8-chlorotheophylline 321,
393
chlorpheniramine 315, 320,
322
chlorpromazine 255, 258
chlorpropham 566
chlorpyrifos 537
chlorpyrifos-methyl 537
cholinesterase 17, 208, 364,
535
cholinesterase inhibitor 208,
384
chromium 637
CI 40
cigarettes 499
cigarette butts 506
cigarette smoke 113
cinerins 425
cis-permethrin 427
cis and trans isomers 425

city gas 91
Cl
–
404
clandestine tablets 233
Claviceps purpurea 225
clemastine 315
clinical analytical toxicology
59
clinical toxicology 59
Clitocybe acromelalga 469
Clitocybe clavipes 469
Clitocybe gibba 469
ClO
–
404
ClO
3
–
404
clomipramine 271, 274,
276
clonazepam 284, 285
clorazepate 285
clordiazepoxide 284
clospirazine 259
cloxacillin 395
clozapine 19
club drugs 247
CO 91, 144

CO
2
poisoning 37
CO
3
2–
404
cocaine 207, 212, 215, 377,
383, 387
cockroaches 431
codeine 195, 196
codeine-TMS 201
codeine glucuronide 204
COHb 91, 92, 144
cold trap 160, 161
collision induced
dissociation 41
color tests 113
combined SIM mode 221
complex partial seizures 351
computer system for
diagnosis 45
conductivity detector 39
Conway microdi usion cell
112, 629
copper 637
Coprinus atramentarius 469
coronary artery dilators 649