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

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

© 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

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

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

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

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

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

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

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