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If the calculated concentration is higher than the ULOQ, it is considered undetermined and labeled as above the quantitation limit (AQL or ALQ).
If the concentration of the analyte in the study sample is BQL, some additional
method development may be necessary to increase the sensitivity of the bioanalysis.
If the concentration of the analyte in the study sample is AQL, the samples may
need to be diluted and reanalyzed to provide measurable concentrations of the analyte.
This is frequently the case in quantitation of TK samples after administration of a high
dose of compound in toxicology studies.
Sample Preparation Only charged analytes can be measured by LC–MS/MS
methodology. Some component of the biological matrix may reduce the ability of
the analytes to become ionized and have to be removed from the biological sample
prior to quantitation. This process is called sample preparation.
There are three most commonly used sample preparation techniques used for
PK sample analysis: (a) protein precipitation; (b) solid ph ase extraction (SPE, either
off-line or on-line); and liquid–liquid extraction (LLE) [137]. All three methods
allow for quantitation of the total (not free) drug and can be automated to use in a highthroughput manner.
PROTEIN PRECIPITATION Protein precipitation is perhaps the most frequently used
method of sample preparation in discovery settings. The concept of the PPX is to
denature and remove matrix components (primarily such abundant proteins as
albumin) by mixing plasma samples with quenching agents, water-miscible chemicals in which plasma proteins are less soluble than analyte.
Acetonitrile is by far the most commonly used quenching agent in discovery
bioanalysis. Other frequently used quenching agents are methanol and ethanol,
although there is a potential risk of transesterification for ester-containing prodrugs.
It is critical to ensure that the analyte of interest is soluble and stable in the final
aqueous-organic mixture formed after protein precipitation by organic-quenching
agents. Incomplete solubility of analytes in the aqueous-organic mixture may be
a source of low recovery of the samples containing high concentrations of the
compounds and their metabolites. Special attention is required for protein precipitation for toxicokinetics sample processing due to higher concentrations of analytes.
Acid-driven protein precipitation (e.g., trichloroacetic acid [TCA]) is a potential
alternative to precipitation by organic solvents in the case of very polar analytes stable
in acidic conditions.
SOLID PHASE EXTRACTION The SPE sample cleanup is based on the retention of the
analyte on a very short reversed-phase chromatographic column. Diluted or quenched
biological samples are typically loaded on the SPE device followed by a wash step
removing the matrix components. The analyte is eluted with stronger chromatographic
solvents. The SPE may require more method development but typically provides
cleaner samples than protein precipitation.
The SPE can be used either off-line (typically in 96-well plates) or online [123,
131, 135].
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LIQUID–LIQUID EXTRACTION LLE of the analytes by nonwater-miscible organic sol-
vents is perhaps most familiar for medicinal chemists. The LLE usually provides
cleaner samples than protein precipitation. However, LLE is usually more laborintensive unless automated.
An accurate and precise bioanalysis of PK samples may have a significant impact
on the quality of PK data and their interpretation.
5.7 ENGINEERING MOLECULES WITH DESIR ED ADME PROFILE
This chapter does not describe how key ADME parameters can be “engineered” to
improve the quality of drug candidates (see Chapter 2 for a more detailed discussion).
There is significant literature that discusses SAR around ADME properties of small
molecules from the “Lipinskis rule-of-five” [138] to some rules of thumbs [139–145].
5.A APPENDICES
These appendices contain the normal physiological values for commonly used
laboratory animal species and man. These data help discovery scientists for a
mechanistic explanation of ADME data. Care shoul d be exercised in using the data.
The data should serve as a guide rather than be used in an absolute sense. Additionally,
these physiological values are being constantly updated and depending on the species
and laboratory conditions, under which they are measured, and can and will differ in
many literature sources.
5.A.1 General Morphinometric Data for Different Species
TABLE 5.A.1 General Morphinometric Data for Various Laboratory Animal Species
Parameter Mouse Rat Rabbit Dog Monkey Human
Species weight (kg) 0.02 0.25 2.5 10 5 70
Surface area (m
2
) 0.008 0.023 0.17 0.51 0.32 1.85
Mean life span potential (years) 2.7 4.7 8.0 20 22 93
Total plasma protein (g/100 mL) 6.2 6.7 5.7 9.0 8.8 7.4
Plasma albumin (g/100 mL) 3.27 3.16 3.87 2.63 4.93 4.18
Plasma a-1 acid glycoprotein
(g/100 mL)
1.25 1.81 0.13 0.37 0.24 0.18
Total ventilation (L/min) 0.025 0.12 0.80 1.50 1.67 7.98
Respiratory rate (min
–1
) 163 85 51 23 38 12
Heart rate (beats per minute) 624 362 213 96 192 65
Oxygen consumption
(mL/(h g) of body weight)
1.59 0.84 0.48 0.34 0.43 0.20
Source: Adapted from Ref. 16.
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5.A.2 Organ Weights in Different Species
TABLE 5.A.3 Relative Weights of Brain Regions in Rat and Human (Percent of Total
Brain Weight)
Region of Brain Rat Human
Cerebrum 51.6 85–88
Cerebellum 14.3 10–12
Midbrain 15.2
Olfactory lobe 2.8
Brain stem 16.1 1.9–2.3
Medulla 11.5
Pons 4.6
Source: Adapted from Ref. 146.
TABLE 5.A.2 Relative Organ Weight (Percent of Body Weight) in Different Species
Mouse Rat Rabbit Dog Monkey
a
Human
b
Organ
Adipose tissue 21.42
Adrenals 0.048 0.019 0.009 0.03 0.02
Bone 10.73 0.36 8.10 14.29
Brain 1.65 0.57 0.78 1.38 2.00
GI tract 3.34 1.71
Stomach 0.6 0.46 0.79 0.21
Small intestine 2.53 1.40 2.22 0.91
Large intestine 1.09 0.84 0.67 0.53
Heart 0.50 0.33 0.20 0.78 0.45 0.47
Kidneys 1.67 0.73 0.52 0.55 0.40 0.44
Liver 5.49 3.66 2.87 3.29 2.39 2.57
Lungs 0.73 0.50 0.82 0.92 0.76
Muscle 38.40 40.43 45.65 40.00
Pancreas 0.32 0.23 0.14
Skin 16.53 19.03 3.71
Spleen 0.35 0.20 0.04 0.27 0.08 0.26
Testes 0.109 0.02
Thymus 0.15 0.02 0.03
Thyroid 0.005 0.01 0.008 0.03
a
Rhesus.
b
From Ref. 147.
Source: Adapted from Ref. 146.
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5.A.3 Organ, Tissue, and Fluid Volumes in Different Species
5.A.4 Blood Content in Different Rat Organs
TABLE 5.A.5 Blood Content in Rat Organs and Tissues
mL Blood/g
Tissue [148]
mL Blood/g
Tissue [149]
Bone 19.1 45
Brain 13.5 11
Fat (dorsal) 4.6
Fat (perirental) 7.9
Gut 10.4
Intestine 28
Heart 61.0 60
(continued )
TABLE 5.A.4 Organ Volumes (mL) and Miscellaneous Volumetric Data in Different
Species
Mouse Rat Rabbit Dog Monkey
a
Human
Body weight (kg) 0.02 0.25 2.5 10 5 70
Brain 2.2 72 1,450
Liver 1.3 19.6 100 480 135 1,690
Kidneys 0.34 3.7 15 60 30 280
Heart 0.095 1.2 6 120 17 310
Spleen 0.1 1.3 1 36 192
Lungs 0.1 2.1 17 120 1,170
Gut 1.5 11.3 120 480 230 1,650
Muscle 10.0 245 1,350 5,530 2,500 35,000
Adipose 10.0 120 10,000
Skin 2.9 40.0 110 500 7,800
Blood 1.7 13.5 165 900 367 5,200
Blood (mL/kg)
b
85 54 66 90 73 74
Plasma 1.0 7.8 110 515 224 3,000
Plasma (mL/kg)
b
50 31 44 51.5 45 43
Hematocrit 0.45 0.46 0.36 0.42 0.41 0.44
Total body water 14.5 167 1,790 6,036 3,465 42,000
Total body water
b
(mL/kg) 725 668 716 604 693 600
Intracellular fluid 92.8 1,165 3,276 2,425 23,800
Intracellular fluid
b
(mL/kg) 371 466 328 485 340
Extracellular fluid 74.2 625 2,760 1,040 18,200
Extracellular fluid
b
(mL/kg) 297 250 276 208 260
a
Rhesus.
b
Calculated based on the values in this table.
Source: Adapted from Refs. [16, 146].
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5.A.5 Biofluid Flow through the Organs in Different Species
TABLE 5.A.6 Blood, Urine, and Bile Flow through the Body Organs in Laboratory
Animals and Humans
Mouse Rat Rabbit Monkey
Dog Human
Body weight (kg) 0.02 0.25 2.5 5 10 70
Blood Flow (mL/min)
Heart 0.28 3.9 16 60 54 240
Cardiac output 8.0 74.0 530 1086 1200 5600
Liver (Q
H
) 1.8 13.8 177 218 309 1450
Hepatic artery 0.35 2.0 37 51 79 300
Portal vein 1.45 9.8 140 167 230 1150
Kidneys 1.3 9.2 80 138 216 1240
Brain 1.3 72 45 700
Spleen 0.09 0.63 9 21 25 77
Gut 1.5 7.5 111 125 216 1100
Muscle 0.91 7.5 155 90 250 750
Adipose 0.4 32 20 35 260
Skin 0.41 5.8 54 100 300
Fluid Flow
Urine flow (mL/day) 1.0 50.0 150 375 300 1400
Bile flow (mL/day) 2.0 22.5 300 125 120 350
GFR 0.28 1.31 7.8 10.4 61.3 125
a
Rhesus.
Source: Adopted from Ref. 16.
TABLE 5.A.5 (Continued )
mL Blood/g
Tissue [148]
mL Blood/g
Tissue [149]
Kidney 45.9 92
Liver 57.2 99
Lungs 175.0 111
Lymph node 8.2
Muscle (skeletal) 4.0 4
Pancreas 32.1
Seminal vesicles 11.0
Skin 2.1 20
Spleen 321.0 86
Stomach 10.8
Testis 7.3 6
Thymus 8.8
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5.A.6 Anatomical Characteristics of GI Tract in Different Species
TABLE 5.A.8 Anatomical Characteristics of the (Small) Intestine for Various Species
Rat (Wistar) Rabbit Dog Monkey Human
Small intestine 0.13 1.51 2.48 6.25
(m) 0.82 4.14
(% of total) 64 60 85 79
Diameter (cm)
Small intestine 0.3–0.5 1 1.2–2 5
Duodenum 2–2.5 1.5–2 3–4
Region Surface Area Relative to Body Area (%)
Duodenum 1.8 10.3
Jejunum 19.8 74.9
Ileum 0.4 22.9
Absorption surface area (m
2
)
Duodenum 0.09
Jejunum 60
Ileum 60
Cecum 0.04 0.44 0.08 0.05–0.06 0.15
(m) 0.06 0.61
(% of total) 31 11 2 2
Colon 0.01 1.23 0.34 0.4–0.5 1.50
(m) 0.26 1.65 0.60
(% of total) 5 28 13 19
Source: Adapted from Ref. 150.
TABLE 5.A.7 Relative Weights (Percent of Total Body Weight) of Various Sections of
the GI Tract
Segment Mouse Rat Dog Human
GI tract 4.22 2.37–3.32 4.40 1.65
Stomach 0.60 0.40–0.52 0.65–0.94 0.21
Fore stomach 0.16 0.13
Glandular stomach 0.44 0.27–0.39
Small intestine 2.53 0.99–1.93 1.61–2.84 0.91
Duodenum 0.15 0.19–0.25
Jejunum/ileum 1.24 1.36–1.46
Large intestine 1.09 0.80–0.89 0.65–0.69 0.53
Cecum 0.30 0.32–0.35
Colon 0.44–0.54 0.23–0.47
Source: Adapted from Ref. 146.
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5.A.7 The pH and Motility of GI Tract in Different Species
5.A.8 Phase I and Phase II Metabolism in Different Species
TABLE 5.A.10 The GI Tract Motility in Laboratory Animal Species
Parameter Mouse Rat Rabbit Dog Monkey Human
Weight (kg) 0.02 0.25 2.5 10 5 70
Transit time (min)
Stomach 96 78
Small intestine 88 110 238
Whole gut 770 2350
Source: Adapted from Refs. [16, 146].
TABLE 5.A.9 Intestinal pH in Different Species
Mouse Rat
(Wistar)
Rabbit Dog Monkey Human
Stomach
Anterior 4.5
a
Posterior 3.1
a
Duodenum: fasted (fed) 6.5–7.1 (6.9) 6.0–8.0 6.2–7.5,
4.5–7.5
5.6–6.0,
7–9
5–7,
5.6–6.4
Jejunum: fasted 6–7
Jejunum/ileum:
fasted (fed)
(7.8)
Ileum: fasted (fed) 7.1
a
7.0, 7.4,
7–8
Cecum: fasted (fed) 6.8 (6.7) 6.6 6.4 5.0 5.9
Colon: fasted (fed) 6.6 (7.1) 7.2 6.5 5.1 5.5–7
Rectum: fasted 7
Feces 6.9
a
7.2
a
6.2
a
5.5
a
a
From Refs. [16, 146] with no specification of meal consumption but most consistent with other data for
fasted state.
Source: Adapted from Refs. [150, 151].
TABLE 5.A.11 Substrate-Specific P450-Mediated Enzymatic Activity (pmol/(min mg)
of protein) in Different Species
Major Human
P450
Dog
(Beagle)
Monkey
(Cynomolgus)
Monkey
(Rhesus) Human
Ethoxyresorfurin
O-deethylation
1A1/1A2 46 25 240 85 295 71 21 14
Coumarin
7-hydroxylation
2A6 75 17 678 209 289 125 448 330
Tolbutamide
4-hydroxylation
2C9/10 50 12 47 988 37
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TABLE 5.A.12 Substrate-Specific P450 Content and Drug Oxidation Activities in Liver
Microsomes of Various Animal Species
Parameter
Major
Human
P450 Rat Dog Monkey Human
Totalprotein in liver (mg) 1,738 123 43,200 4,199 7,875 222,700
P450 content (pmole
P450/mg protein)
673 50 385 36 1,030 106 307 160
Phenacetin
O-deethylation
(pmole/(min mg)
protein)
1A2 36 20 28 10 110 20 32 30
Coumarin
7-hydroxylation
(pmole/(min mg)
protein)
2A6 <112 5 209 126 21 22
Pentoxyresorufin
O-dealkylation
(pmole/(min mg)
protein)
2B6 70 10 60 10 35 55 5
Phenytoin
p-hydroxylation
(pmole/(min mg)
protein)
2C9 44 884 645 12 29 15
Mephenytoin
4-hydroxylation
(pmole/(min mg)
protein)
2C19 74 15 106 13 144 26 39 23
(continued )
TABLE 5.A.11 (Continued )
Major Human
P450
Dog
(Beagle)
Monkey
(Cynomolgus)
Monkey
(Rhesus) Human
S-mephentoin
4
0
-hydroxylation
2C19 12.7 0.4 106 50 30 744 30
Bufuralol
1
0
-hydroxylation
2D6 49 11 530 154 558 189 34 20
N-nitroso
dimethylamine
N-demethylation
2E1 694 220 758 286 610 76 761 319
Erythromycin
N-demethylation
3A 876 316 2949 298 1997 437 153 74
Midazolam
1
0
-hydroxylation
3A 1053 257 1330 174 1107 326 320 182
Source: Adapted from Ref. 150.
APPENDICES
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TABLE 5.A.12 (Continued )
Parameter
Major
Human
P450 Rat Dog Monkey Human
Bufuralol
1-hydroxylation
(pmole/(min mg)
protein)
2D6 743 74 12 2 471 58 20 14
Aniline p-hydroxylation
(pmole/(min mg)
protein)
2E1 403 57 280 91 227 165 696 986
Benzphetamine
N-demethylation
(pmole/(min mg)
protein)
3A/2B 2,387 639 348 78 940 200 544 427
Ethylmorphine
N-demethylation
(pmole/(min mg)
protein)
3A/2B 3,370 621 273 149 1,080 249 423 543
Erythromycin
N-demethylation
(pmole/(min mg)
protein)
3A4/5 930 164 144 70 586 101 244 212
Nifedipine oxidation
(pmole/(min mg)
protein)
3A4/5 820 187 456 126 3,826 131 604 691
Source: Adapted from Ref. 150.
TABLE 5.A.13 Phase II Enzyme Activities in the Liver of Various Animal Species
Phase II Enzyme Activity
(pmole/(min mg)) Dog
Monkey
(Cynomolgus)
Monkey
(Rhesus) Human
Acetaminophen glucuronyl
transferase
407 111 489 93 625 267 78 33
17a-Ethynyl estradiol
glucuronyltransferase
142 55 62 32 95 19 85 42
Acetaminophen
sulfotransferase
176 36 347 22 360 50 88 28
17a-Ethynyl estradiol
sulfotransferase
91 19 44 14 43 16 39 12
6-Mercaptopurine methylase 1.9 0.5 4.1 1.2 3.3 0.6 3.5 0.8
3, 4-Dicholoronitrobenzene
glutathione S-transferase
(nmol/(min mg))
7280 565 2820 560 2720 430 1070 130
Source: Adapted from Ref. 150.
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ACKNOWLEDGMENTS
The authors would like to acknowledge our colleagues who by many means (valuable
recommendations, checking the accuracy of the information, supplying useful
references, and proof reading) improved the quality of this chapter.
Pfizer colleagues: Rajesh V. Devraj, Heather Dowty,Tim Heath, Kathy Hotz Kathy
Mandrell, Dean Messing, Scott Obach, Denise K. Pretzer, YuriyPyatkivskyy, Charles
Stankovic, Joseph W. Strohbach, Jay Wendling, Steve Wene; and MPI Research
colleagues: Travis Devlin and Edith M. Luckett.
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