British Journal of Clinical Pharmacology
A descriptive systematic review of salivary TDM in neonates and infants.
Authors: Laura Hutchinsona MPharm, Marlene Sinclaira* PhD, Bernadette Reida PhD,
Kathryn Burnettb PhD, and Bridgeen Callanb* PhD.
a. Institute of Nursing and Health Research, Ulster University
b. Biomedical Sciences Research, Ulster University
*address for correspondence:
Bridgeen Callan
School of Pharmacy and Pharmaceutical Sciences
Biomedical Sciences Research Institute
Ulster University
Cromore Road
Coleraine
Co. Londonderry
Telephone: 0044 28 7012 3510
Email: [email protected]
Marlene Sinclair
School of Nursing
Institute of Nursing and Health Research
Ulster University
Newtownabbey
Co Antrim
BT37 OQB
Telephone: 0044 28 9036 8118
Email:[email protected]
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Sources of support: Department for the economy (DfE), Northern Ireland
Conflict of interest: The views expressed in this manuscript are those of the authors and
do not reflect that of the Department of the Economy. The author has no conflicts of interest.
Word Count: 6,186
Table 1. Biological Matrices Used in infants for TDM-Advantages and Disadvantages
Table 2. Compounds for infant and neonate TDM in saliva
Table 3. Acidic Drugs assessed for salivary TDM in infants and neonates
Table 4. Basic Drugs assessed for salivary TDM in infants and neonates
Table 5. Neutral Drugs assessed for salivary TDM in infants and neonates
Figure 1. Medline search strategy
Figure 2 Comparison of mean R values for the 13 compounds assessed
Keywords: Therapeutic Drug monitoring (TDM), saliva, physicochemical properties, pharmacokinetic parameters, infants, paediatric, systematic review
Glossary of Abbreviations
Abbreviation Full meaning
CBZ carbamazepine
CBZ-E carbamazepine-10,11-epoxide
EMIT Enzyme Multiplied Immunoassay Technique
FPIA Fluorescence polarisation immunoassay
GC MS Gas Chromatography Mass Spectroscopy
GFR Glomerular filtration rate
GLC gas liquid chromatography
HPLC High Pressure Liquid Chromatography
HPLC-UV High Pressure Liquid Chromatography-Ultraviolet
IV Intravenous
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LC-MS/MS Liquid Chromatography-Mass Spec/Mass Spec
MDH 10,11-dihydro-10-hydroxy-carbazepine,
MS Mass Spectroscopy
OXC oxcarbazepine
P/S Plasma/Saliva Ratio
S/P Saliva/Plasma ratio
R correlation coefficient
TDM Therapeutic Drug Monitoring
Summary
Introduction
Saliva, as a matrix, offers many benefits over blood in therapeutic drug monitoring (TDM), in
particular for infantile TDM. However, the accuracy of salivary TDM in infants remains an
area of debate. This review explored the accuracy, applicability and advantages of using
saliva TDM in infants and neonates.
Methods
Databases were searched up to and including September 2016. Studies were included
based on PICO as follows: P: Infants and neonates being treated with any medication, I:
Salivary Therapeutic Drug Monitoring vs C: Traditional methods and O: accuracy,
advantages/disadvantages and applicability to practice. Compounds were assessed by their
physicochemical and pharmacokinetic properties, as well as published quantitative saliva
monitoring data.
Results
Twenty-four studies and their respective 13 compounds were investigated. Four neutral
and two acidic compounds, oxcarbazepine, primidone, fluconazole, busulfan, theophylline
and phenytoin displayed excellent /very good correlation between blood plasma and saliva.
Lamotrigine was the only basic compound to show excellent correlation with morphine
exhibiting no correlation between saliva and blood plasma. Furthermore, any compound with
a pKa within physiological range (pH 6 – 8) gave a more varied response.
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Conclusion
There is significant potential for infantile saliva testing and in particular, for neutral and
weakly acidic compounds. Of the properties investigated, pKa was the most influential with
both logP and protein binding having little effect on this correlation. To conclude any
compound with a pKa within physiological range (pH 6 – 8) should be considered with extra
care, with the extraction and analysis method examined and optimized on a case-by-case
basis.
Introduction
Therapeutic drug monitoring (TDM) is defined as “measuring serum concentrations of a drug
in a single or multiple time points in a biological matrix after a dosage” [1]. It is especially
useful when the relationship between drug plasma concentration and effect is stronger than
between drug dosage and effect, and is vital when measuring drugs with a narrow
therapeutic range [1, 2] or if concentrations resulting from a given dose are unpredictable
due to high inter/ intra patient variability [3]. Pharmacodynamics and pharmacokinetic factors
such as absorption, distribution, metabolism and excretion lead to individual variability [2].
This is especially true in young children, who, for example, have a higher rate of
development of these processes, which is greatest from birth-1 month but changes also
occur during infancy and again during childhood [4, 5, 2, 6]. They tend to have lower protein
binding which increases the free fraction of drugs and a less mature glomerular filtration rate
(GFR) [7] which means drugs may be retained for longer in those <6 months old, while
glucuronidation may not reach adult values until 3-6 months old [5]. It is therefore not
surprising that validated dosage regimens are limited in this population, with approximately
70% of drugs prescribed to children and >93% prescribed to critically ill neonates being done
so off label [8, 9] .
The use of serum/plasma in TDM, is well established and the most clinically used method
[10,11]. Nevertheless, extensive blood sampling in infants is not practical, or ethical due to
pain, anxiety and risk of infection [12]. There are a significant number of validated matrices
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available for TDM in adults,[11] [13, 14] however, these are not always appropriate for TDM
in infants or neonates, for example tears, sweat and hair due to lack of availability. Table 1
reflects the available matrices for TDM in infants and outlines the advantages and
disadvantages of each.
Technological advances in TDM in the past four decades have led to saliva testing becoming
more popular in the diagnosis of diseases, disease progression and detection of drugs [15,
16]. It has become a valuable clinical method due to its non-invasive nature, no need for
specialist training or equipment, patient preference and the ability to compare easily with
plasma concentrations [16, 17]. This is because most compounds found in blood are also
present in saliva. In infants this method seems optimal to prevent distress and ensure patient
safety [18]. If there is a consistent comparison between saliva and plasma, and if the
biological response to the drug is proportionate to its plasma concentration, then salivary
concentrations may provide a valuable measurement of TDM.
Several studies have been conducted in adults in regards to saliva TDM, with it being a
proven accurate method in relation to many drugs including artemisinin [20], digoxin [21],
lamotrigine [22] and a number of other antiepileptic drugs [23]. However, these reference
ranges do not automatically transfer to infants, and the accuracy of salivary TDM in infants is
still an area of debate [24, 25] , with confusion between collection methods and simulation
verses no stimulation
The number of studies looking at TDM involving infants is low primarily due to poor consent
rates, limited blood availability, low volume drug concentration assays and a lack of
expertise in this area [26]. Studies in paediatrics are usually disease orientated and
expensive as drug companies are unable to recover costs in this limited population [27].
Consequently, there is a need for novel and accurate study designs which reduce these
barriers: saliva TDM may meet these requirements.
This review aims to explore the accuracy, applicability and advantages of using saliva TDM
in infants and neonates.
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Methods
The review question was defined using PICO as follows: P: Infants and neonates being
treated with any medication. An Infant was defined by Medline as a child between 1 and 23
months of age [28] and a neonate as an infant during the first 28 days after birth. For the
purpose of this review the term infant has been used to include all those <24 months I:
Salivary TDM vs C: Traditional methods and O: accuracy, advantages/disadvantages and
applicability to practice.
Search methods for identification of studies: A search strategy was defined with the help
of the subject librarian and was adapted according to each database. Five databases:
Medline, Embase, CINAHL, PubMed and Scopus were searched, along with The Cochrane
Library and Google Scholar up to and including September 2016. There were 321 potential
studies, following removal of duplicates and screening by two members of the research team
by title and abstract, 24 were eligible for full paper review. In all cases the primary sources
were used unless no subsequent peer reviewed article was published. Review articles of
importance were reviewed. The search terms used in Medline are detailed in Figure 1. The
search terms used included therapeutic drug monitoring, saliva*, infant and their numerous
variations. We also scrutinised reference lists for those which met the criteria.
The saliva/plasma concentration ratio (S/P) is a very useful parameter when assessing
drugs contained within the saliva. This ratio is determined by a number of physiological
factors which affect the passage of drug from blood to saliva. These include the pH of the
oral fluid and plasma, the saliva flow rate and the pathophysiology of the oral cavity [51].
With the exception of salivary pH, most of these remain constant. The primary drug
properties which also play a key role in the passage of drugs into saliva include the pKa of
the drug, its molecular weight, spatial configuration and lipid solubility of the analyte and
degree of protein binding in plasma and saliva [15]. Once identified, compounds were
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assessed by their physicochemical and pharmacokinetic properties, as well as published
quantitative saliva monitoring data.
Results
Thirteen different compounds were considered suitable for inclusion within this review. Of
the compounds chosen, they were categorised as being acidic (n=4), basic (n=3) or neutral
(n=6). In the case of an amphoteric compound, such as morphine, it was included within the
category to which it has the highest ionisation potential, in this case basic. Acidic compounds
were deemed to have a pKa less than or equal to 10, and basic compounds were
considered as having a pKa greater than or equal to 4.0. Any pKa value outside of this range
(whether acidic or basic) would not be substantially affected by the physiological pH range of
saliva (≤ 0.1% ionised) and was thereby categorised as neutral. Table 2 displays the
compounds assessed within this review and includes the chemical structure, indications and
both the physicochemical and pharmacokinetic parameters.
As the various authors have described the correlation between the experimental blood
plasma levels of a compound and that of saliva levels in different manners, for the purpose
of this review, the correlation R value has been categorised as either excellent, very good,
good, fair or poor. Tables 3 - 5 summarise the information on each of the thirteen
compounds.
Acidic compounds (pKa ≤ 10)
Four acidic compounds have previously been studied for saliva content in infants (Table 2)
with the pKa ranging from digoxin (7.15) to theophylline (8.81). The individual compounds
are discussed below and detailed in Table 3.
Theophylline
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Five studies were included within this review on TDM of theophylline in infants [29, 30, 31,
32, 24] . Of these studies four were deemed to have excellent correlation [29, 30, 31, 32]
(R=0.97 p<0.01). Of these four studies, two used stimulation to enhance saliva collection,
one did not use stimulation and the other collected both stimulated and unstimulated saliva
for comparison. There was no difference in correlation in any of these studies by the
introduction of stimulation to increase saliva production.
The earliest study to be included for the monitoring of theophylline was published in 1980 by
Khanna et al. [24] assessing 10 premature neonates This was the only study to conclude
only a fair correlation between the blood and saliva monitoring. In contrast to the Toback [29]
study just three years later Khanna reports a correlation value as low as 0.7 compared to
Toback’s 0.98. Both studies were similar in their use of HPLC for analysis, no stimulation, a
similar time period for sample collection following IV administration and assessed a similar
amount of premature neonates. The only difference appears to be storage method with the
Khanna study freezing samples until analysis (no further details given) whereas the Toback
study either stored samples at 4ᴼC for 48 hours or analysed immediately.
The method chosen for analysis (Table 3) also varied between the five studies to include; in
the most recent study (2007) by Chereches-Panta et al. [32] assessing 13 infants Gas
Chromatography Mass Spectroscopy (GC MS) was utilised. MS was used in the 2001 study
by Culea et al [31] with 13 neonates whereas Enzyme Multiplied Immunoassay Technique
(EMIT) was used in the 1990 study by Siegel et al. [30] with 31 children and High Pressure
Liquid Chromatography (HPLC) by Toback et al [29] in 1983 and Khanna in 1980.
One further point of interest when comparing these five studies, is that only four of the five
describe the S/P ratio, which varies significantly between the studies, ranging from 0.69 [32]
to 1.53 [31]. When considering theophylline, overall stimulated saliva sampling is thought to
be an accurate method of TDM while treating levels >8µg/ml with caution [30] and
consideration needs to be given to the higher saliva/plasma (S/P) theophylline concentration
ratio in neonates as compared to children and adults. The S/P ratio of 1.24 in Khanna’s
study was similar to the observations of others [33, 34],while Toback et al [29] suggest the
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ratio to be closer to 1. Also, that found by Culea [31] is low, although they found the ratio
higher in the infants than children. The lower correlations with unstimulated saliva which
were seen by Siegel et al [30] were thought to be due to greater variability in the theophylline
saliva concentration due to changes in flow rates and pH. However, it has been determined
that the secretion of theophylline is not influenced by these factors [32, 35, 33, 36]. The
difference seen between the populations could be due to differences in protein binding in
these groups, but this requires further exploration. It has, however, been suggested that
neonates have minimal protein binding of theophylline [37] and that proteins in this group
generally differ from that found in children and adults [38, 39]. In addition, high plasma
concentrations of free fatty acids, unconjugated bilirubin and steroids in neonates can
compete with certain drugs for binding sites. It has also been suggested that pH changes in
blood as little as 0.2 may affect the protein binding [40].
With the exception of the 1980 study, all other studies were in agreement that theophylline
shows excellent correlation between blood and saliva, regardless of whether stimulation is
used and independent on analytical method indicating that saliva TDM is useful for this
compound.
Phenytoin
There were four studies published assessing phenytoin in infant and children’s saliva which
were included within this article. The studies all took place in the last century; 1975 [41],
1981 [42, 43] and 1990 [44]. The correlation seen between the samples and indeed
between the different studies was excellent - very good. Similar to the theophylline study a
range of different analytical methods were employed to quantify the samples. In the 1981
study by Goldsmith and Ouvrier [42] a large study was carried out using two different assay
methods, gas liquid chromatography (GLC) and EMIT assessing 202 children between the
ages of 5 months and 18 years. They report that the GLC assay had very good correlation
(R=0.94) whereas the EMIT was not as significant (R=0.86). They also describe better
correlation with fresh samples compared to frozen ones.
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In the most recent study on phenytoin (1990) by Lifschitz et al. [44] , 16 children and infants
taking oral phenytoin were studied. Stimulated saliva and blood were analysed by
fluorescence polarisation immunoassay (FPIA) and the correlation between blood and saliva
described as excellent (0.99). When comparing the S/P ratio for all four studies, they are in
close agreement to one another, with an average value of 0.11 ± 0.013.
Phenytoin in adults and older children has shown wide individual and marked interpatient
variability, including age-dependent metabolism [45], although it has a well-documented S/P
ratio between 0.10-0.12 [23, 41, 46, 47, 48, 49], which is in agreement with all of these
studies [41, 42] . All studies showed high correlations meaning TDM using saliva is an
accurate method for phenytoin, however, as there is a wide interpatient metabolism
associated with it, lack of correlation between drug dose and concentration needs taken into
account. The use of stimulated/unstimulated saliva seems to have little effect on correlation
and researchers should use EMIT with caution.
Phenobarbital
Four published studies were included within this review [41, 42, 43, 50]. Each of these
studies had assessed phenobarbital in saliva in infants combined with that of other
anticonvulsants such as phenytoin, described above. The correlation of saliva to blood
varied significantly between studies, ranging from excellent as described by Cook et al. [41]
where unstimulated saliva and plasma were analysed by radioimmunoassay (R=0.98
p<0.001) in a study assessing 38 patients between 8 months and 62 yrs old. In stark
contrast to this, the 1981 study described by Mucklow et al. [43] assessing 19 infants, was
unable to identify a linear relationship between dosage and saliva concentration for
phenobarbital at all. Goldsmith and Ouvrier [42] found a very good correlation in 15 infants
with a S/P ratio=0.32 +/- 0.06 and no significant difference was seen between the different
age groups that were studied. For the GLC assay the correlation was very good (R=0.94)
and the EMIT was similar (R=0.92). A later study by Gorodischer et al. in 1997 [50] studied
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stimulated saliva and plasma which was analysed by HPLC found the correlation coefficient
to be poor (R=0.65). However, those >8 years had the highest correlation (R=0.93).
The S/P ratio was given in three of the four studies, and found to be in good agreement with
one another, with an average ratio of 0.28 ± 0.06. The S/P ratio described by Cook et al [41]
and Goldsmith et al [42] is similar to that found in adults which varied between 0.30-0.37
[41, 46, 47, 48, 49] [54, 55].
.
Therefore, salivary TDM of phenobarbital has the potential to be an accurate method in
infants. However, the age of the patient must be considered, as too should stimulation as the
two studies, which displayed excellent /very good correlation, neither of which used
stimulation. However, a study by Knotts which included stimulation in adults and older
children reported excellent correlation (R=0.95) [53]. The patients in this study were older
and this was the only major methodological difference, meaning that salivary excretion of
phenobarbital may be regulated differently in older and younger children which is in line with
this study when they looked at ages of the patients. Those >8 years had the best correlation
(R=0.93 plasma total and 0.93 Plasma free).
Digoxin
In the three studies included within this article, the correlation varies from good [25, 58] to
fair [59] . As seen in the 1998 study by Berkovitch et al. [25] good correlation (R=0.87) in 11
children (9 of which were infants) receiving oral digoxin was presented. Plasma and saliva
was analysed using FPIA. A further study in 2003 by Zalzstein et al. [58] also showed a good
correlation (R=0.83 p<0.01) between stimulated saliva and serum in 18 children (again 9 of
which were infants) receiving oral digoxin. The samples were also analysed using FPIA,
however variability existed in individual S/P concentration ratios. Low plasma concentrations
correlated with negligible or undetectable saliva concentrations. No plasma levels were
above the agreed toxic range of 2ng/ml, however, one saliva was deemed toxic, without the
plasma following suit. In three saliva samples the digoxin concentration was not detectable
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even though the plasma levels were within therapeutic range. An earlier study by Krivoy et
al. conducted in 1981 [59] compared plasma with unstimulated saliva using the RIA digoxin
kit. A S/P ratio of 0.66 +/- 0.20 and a fair correlation co-efficient (R=0.71 p<0.001) was
noted, however, it showed a wide range in the ratios. On comparison between the S/P ratios
quoted they are vastly different in the two studies that present them.
Digoxin is an acidic drug with a pKa of 7.15, meaning that % ionisation will vary significantly
with small changes in physiological pH. An increase in pH from 6 to 7 will see the % of
digoxin ionised increase from 6.6 to 41.45%. Krivoy et al [59] stated that saliva was an
accurate method of TDM, however, the poor correlation coefficient does not reflect this. In
contrast, Berkovitch reported a much higher correlation and stated it was not a reliable
method. They thought it was possible that digoxin did not enter saliva by diffusion alone, but
also by active secretion and that there was a possibility of endogenous digoxin-like
substances present in the saliva [6]. Krivoy et al [59] found similar correlations to Berkovitch,
showing the stimulation of saliva yields better correlation, yet further studies will need
conducted to determine if, like in adults, TDM using saliva is an accurate method in infants
when no stimulation is used. There is also a need for a standardised method of sample
collection, storage and analysis before a ratio can be accurately determined as high
variability is demonstrated in these studies.
Basic compounds (pKa ≥ 4)
Three basic compounds have previously been studied for saliva content in infants (Table 2)
with the pKa ranging from gentamicin (10.18) to lamotrigine (5.87). The individual
compounds are discussed below. Unlike with the acidic compounds where each compound
had more than two studies to allow comparison, the number of infant studies for TDM in
basic drugs is much more limited with only four studies in total for the three compounds
(Table 4).
Gentamycin
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Only one study satisfied conditions for inclusion within this review. In the 2000 study by
Berkovitch et al. [60], serum and stimulated saliva in 10 infants were analysed using FPIA. A
good correlation was noticed in once daily dosing patients (R=0.89 p<0.0001) but no S/P
ratio was determined. Gentamicin is a hydrophilic drug and with a pKa of 10.18 and will
therefore be positively charged at physiological pH [60], hence, although it is only weakly
protein bound, it would be expected that it does not appear in saliva as Mahmod et al. [61]
states. Berkovitch did find levels in saliva, however, [25] they found no correlation when the
dose was three times a day, it was thought that it must take longer for the ionised molecules
to enter and equilibrate with saliva and hence once daily dosing allows such a time lag as
the measurements are taken 24 hours after dosing. This study suggests it is a reliable and
accurate method to monitor trough levels in infants on a once daily dosing regimen,
however, the lack of studies on this drug leave the results unreliable and more studies need
conducted before an accurate representation of the S/P ratio can be determined
Morphine
In one 1997 study by Kopecky et al. [62] 15 children receiving IV infusions of morphine were
analysed using a solid phase serum morphine radioimmunoassay. The S/P ratio was 2.28
+/- 2.84. There was no correlation between saliva and plasma (R=0.04 p=0.89). Because
citric acid decreased the salivary pH the authors examined the analytical effect of different
pH values on the morphine concentrations in saliva across a pH range of 3.96-8.06 and
there was no difference in assay performance. This study would suggest that determination
of morphine levels from saliva cannot be used as a quantitative tool to predict serum
concentrations, although further studies are required to confirm these findings.
Lamotrigine
Two studies for lamotrigine where found to include within this review. A study by Ryan et al.
in 2003 [63] studied unstimulated saliva and serum using HPLC-UV analysis. The
correlations were tested over two laboratories (R=0.81 and R=0.86). The S/P ratio was 0.62
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+/- 0.19. They found the correlation to be excellent in children (R=0.94) and that the S/P ratio
was less (0.56) than in adults however, these values was based on only one infant.
In a later study (2005) conducted by Malone et al, [19] stimulated and unstimulated saliva
along with serum were studied in an adult group and in 20 children (1 – 16 years) using
HPLC analysis. The data in adults showed that within the first two hours following oral
administration, the results had a wide scatter, after exclusion of these the results they
showed an excellent correlation (R=0.9841 p<0.0001) (n=98). The S/P ratio was lower in the
adults than children (41.7 +/- 7.07% for unstimulated saliva vs 42.1 +/- 6.52% for stimulated).
In children the ratio was 47.6 +/-7.2% and 46.7 +/-6.2%. There was a close correlation
between the concentrations of lamotrigine in stimulated and unstimulated saliva, with the
mean S/P ratio=0.49. As plasma lamotrigine concentrations increased from 1mg/L to
10mg/L, the mean saliva to plasma ratio changed from 41.8% to 48.8%. Saliva TDM of
lamotrigine can be an accurate and reliable method, although it must be ensured that no oral
contamination occurs [63], It should be noted that Malone et al [19] found that as plasma
lamotrigine concentrations increased from 1mg/L to 10mg/L, the mean saliva to plasma ratio
changed from 41.8% to 48.8%, which was statistically significant indicating that saliva to
plasma lamotrigine ratios are concentration-dependent. They believe this may be due to
plasma protein binding saturation as the drug’s plasma concentration rises.
Neutral compounds
Six neutral compounds have been included within this review. They have been classified as
neutral if they have no ionisable functionality or if their pKa value is such that a unit change
in physiological pH would have less than a 0.1 variation in % ionisation, Table 2. This is
further classified as a basic pKa less than 4 and an acidic pKa greater than 10. The
individual compounds are discussed below with detailed criteria presented in Table 5.
Busulfan
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The one study included within this article suggests that TDM of busulfan in unstimulated
saliva is a valid alternative to plasma sampling in infants with excellent correlation (R=0.958)
[64]. Rauh et al [64] demonstrated that busulfan in saliva was stable for up to 48 hours at
4oC (concentration decrease <5%). Analysis was conducted using LC-MS/MS also allowing
for high output sample analysis. The S/P ratio was 1.09. Their saliva collection method,
however, would not be suitable for all infants as some do not actually take a pacifier. The
excellent linear regression makes this drug look ideal for TDM using salivary samples in this
population, however only one infant was involved in the study conducted with 10 children
and therefore further work is required to support the findings.
Fluconazole
One study has been conducted on fluconazole TDM using unstimulated saliva compared
with serum in paediatric patients [65]. The study involved 19 children, 10 of which were
infants, and analysis was again by LC-MS-MS. The correlation was excellent (R=0.96 p<0.1)
and the S/P ratio was 1.0. Van der Elst et al [65] describes how at high concentrations of
fluconazole, the amount in saliva was less than in serum, suggesting saturation in oral fluid.
The S/P drug concentration ratio did not significantly differ in patients receiving oral
treatment versus IV (P = 0.791)
Salivary fluconazole was shown to be stable for 17 days at room temperature giving more
evidence to the potential for home monitoring [65].
Caffeine
Together with theophylline, caffeine had the highest amount of studies included within this
systematic review, with each compound having five studies on TDM using saliva in infants.
The largest of these studies was carried out in 2001 by De Wildt et al. [66], whereby 140
premature neonates were given IV caffeine. Their saliva was stimulated immediately before
collection, 5-10 minutes before collection and unstimulated saliva was also collected.
Plasma and saliva were analysed using HPLC with UV detection. The unstimulated and
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stimulated immediately before the collection groups were not well correlated. The citric acid
stimulated 5-10 minutes before collection had the strongest correlation (R=0.89) with little
variability. In conclusion, they determined that the S/P ratio was 0.7and that this method was
reproducible and feasible when saliva was stimulated 5-10 minutes before collection. An
earlier study was conducted by Khanna et al. [24] on 7 premature infants who received
caffeine via an orogastric tube. Serum and unstimulated saliva was analysed using straight
phase HPLC. A good correlation (R=0.84 +/-2.8 µg/ml) was seen. The P/S ratio was
determined at 1.40, however if we consider the S/P ratio, which has been quoted in the
majority of other articles, then it is identical to that given by De Wildt at 0.7. In this study,
total methylxantine concentration was also measured as caffeine can convert to theophylline
and vice versa [67]. In this case R=0.81 +/-3.3 µg/ml. With the exception of one patient,
when the total concentration in saliva was less than 8 µg/ml, the total in serum did not
exceed 15 µg/ml.
In contrast to this and in a much later study, Lee et al. [68] administered IV caffeine over 7
days to 59 premature neonates. Unstimulated saliva was compared with serum using HPLC
analysis. They used the assessment of precision and bias between two methods and
determined that there was no significant difference in precision between the serum and
salivary data. They concluded that saliva can be used instead of serum to monitor caffeine at
practically any concentration of caffeine even in high maintenance doses of 30mg/kg/day. A
study by Chioukh et al. [69], demonstrated that saliva caffeine levels were comparable to
plasma in 13 infants receiving IV caffeine. The trough caffeine levels were determined from
saliva and serum and analysed by EMIT with a fair correlation (R=0.76). The most recent
study for TDM of caffeine in infants was published in 2016 by Dobson et al. [70]. This study
included 29 premature neonates. Plasma and unstimulated saliva were analysed by HPLC.
They showed good correlation between the salivary and plasma caffeine concentrations over
different doses (R=0.87, p<0.001).
When considering caffeine, the use of saliva for TDM has been shown to have conflicting
results and with major differences in collection methods, use of stimulation and different
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statistical analysis, it is difficult to determine the usefulness of saliva as a method of TDM for
caffeine. In theory this should be an accurate method and if a more robust collection and
analysis method is determined, could prove to be a very useful in this patient group. Lee et
al [68] suggests that the transport of caffeine from blood to saliva is independent of
concentration, meaning it is likely that free caffeine enters salivary ducts by passive diffusion
and not via capacity-limited active transport processes that other drugs may use. De Wilt
[66] found the unstimulated and stimulated immediately before the collection groups were
not well correlated, and it was noted that in more than half of the samples less than 50µl of
saliva was collected which may have been a contributing factor to the weak correlation and
wide variability. Also, applying the citric acid 5 minutes before collection prevented dilution
of the sample by citric acid.
Carbamazepine
Three studies on carbamazepine were included within this review. The earliest of which was
published in 1981 by Goldsmith et al. [42]. They collected unstimulated saliva and plasma
from 202 children (15 of which were infants) and analysed it using GLC and EMIT. They
found that there was better correlation with fresh samples compared to frozen ones. A good
correlation was found in the 15 infants with an S/P ratio of 0.26 and no significant difference
was seen between the age groups. For the GLC assay the correlation was excellent
(R=0.91) and the EMIT was not as significant (R=0.83).
In a later study by Gorodischer et al (1997) [50] 85 children were studied. They collected
stimulated saliva and plasma and analysed them by HPLC. Good correlation was seen
(R=0.84 p<0.001) which was similar to that seen in the EMIT assay by Goldsmith.
Chee et al. published a study in 1993 [71] where they compared carbamazepine and its
active metabolite carbamazepine-10,11-epoxide levels in plasma and unstimulated saliva
samples in 39 children. Analysis was conducted using HPLC. Both CBZ and CBZ-E
concentrations had excellent correlation (R=0.99 and 0.98 respectively) and storage in a
domestic freezer for 1 month had no significant effect on correlation. They believed no oral
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contamination occurred despite not rinsing the participants’ mouths with water. The S/P ratio
is only provided by Goldsmith et al. [42] as 0.26, it has not been quoted in the other studies.
Salivary concentrations of carbamazepine have been shown to correlate significantly with
plasma total and unbound concentrations in children [23, 52, 53, 72] and also in adults [56]
[57, 73, 74], however ratios have been seen to vary widely 0.24-0.37 [23, 46, 49, 74, 75].
The ratio from Goldsmith et al [42] agrees well with three of these studies, two of which [72,
76] were conducted in children. Rylance et al (1979) [76] found that even though a patient is
in so called steady state, carbamazepine levels in saliva may change by 100% during the
course of the day, probably due to its short half-life in children, which also helps explain the
wide variety of ratios seen in studies. Furthermore, EMIT analysis may have a cross reaction
with carbamazepine-10,11 epoxide as mentioned by Bartels et al [72], which may explain the
lower correlation seen in Goldsmith’s study [42]. Overall, unstimulated saliva in infants was
shown to yield better correlations and a standardisation of techniques and assay methods is
needed to confirm these findings.
Oxcarbazepine
In a recent study (2016) conducted by Rui-Rui Li et al. [77] the unstimulated saliva and
plasma 10,11-dihydro-10-hydroxy-carbazepine (MDH) concentrations in 47 children (7 of
which were infants) were compared using HPLC analysis. There was a very good correlation
between saliva and plasma concentrations (R=0.908) with the S/P ratio being 0.71. There
was no significant correlation with patient age or gender and there was no significant
difference with age, OXC daily dose, plasma and saliva MHD concentrations and S/P ratio
when OXC was given in conjunction with other AEDs. No other studies on oxcarbazepine
were included in this review.
Primidone
In a 1981 study by Goldsmith et al. [42] unstimulated saliva and plasma were collected from
202 children and analysed using GLC and EMIT. The S/P ratio was 1.04 for GC and 0.95 for
EMIT. No significant difference was seen between the age groups studied. For the GLC
assay the correlation was very good (R=0.94) and the EMIT was excellent (R=0.98), this
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same study analysed the concentrations of carbamazepine, and found the opposite trend
with analysis methods, in that the GLC was considered to be the better method for
carbamazepine. This corresponds well to adult studies describing the S/P ratios between
0.75-1.08 [46, 47, 51,78].The authors felt the very short half-life of primidone may have
contributed to the greater variation in S/P ratio. However, this single study is too limited to
give an accurate representation of the accuracy of saliva TDM for primidone in infants,
further studies looking into the use of stimulation and collection methods are required.
Discussion
There is a significant amount of evidence available to suggest that saliva is indeed a useful
matrix for TDM in infants (Tables 4-6). Figure 2 displays the average R value achieved from
each of the thirteen compounds analysed. Morphine was the only compound to show
negligible correlation between blood and saliva samples, with all twelve remaining
compounds displaying at least good correlation between saliva and plasma (R>0.8). In
general, the neutral compounds displayed the best correlation between saliva and plasma
concentrations with three neutral compounds (busulfan, fluconazole and primidone)
displaying an R value of greater than 0.95. Acidic drugs also displayed very good correlation,
in particular weakly acidic drugs with a pKa > 8.3. It is perhaps not surprising that the neutral
and very weakly acidic compounds perform better for saliva TDM as derivations in saliva pH
would have no effect on the % ionization of the compounds. There is less evidence available
regarding basic compounds for this method of analysis. However, of the three compounds
included within this review, they display a lower correlation value when compared to either
the acidic or neutral compounds. Of those compounds studied, only one basic compound,
lamotrigine, had a pKa less affected by physiological pH (pKa 5.87) which would be
predominately unionised at physiological pH. Lamotrigine gave excellent potential for saliva
TDM, Figure 2. From analysis of the physicochemical parameters for the compounds it
would suggest that both protein binding and logP had little effect on the correlation. Some
highly protein bound compounds such as phenytoin (90 % protein bound) displayed a
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reduced S/P ratio (0.1) and displayed excellent correlation, likewise primidone which is 70%
protein bound did not have a reduced S/P ratio (1.1) and still recorded excellent correlation.
Additionally, fluconazole with a relatively low protein binding (30%) has an S/P ratio of 1.
Compounds with a pKa within physiological range, i.e. acidic drugs with a pKa < 7.2 or basic
drugs with a pKa > 6 had less correlation, most likely due to variation in % ionization with
variation in saliva pH. The most variation in correlation between different studies of the
same compound was seen from those compounds with a pKa close to 7 (digoxin pKa 7.15
and phenobarbital pKa 7.3) where correlation ranged from excellent to poor (Tables 4-6),
thus suggesting that the pH of saliva is significantly affecting the reliability of these
compounds for analysis via this method. When considering a compound for this type of
TDM, the physicochemical properties of the drug must be taken in to consideration and
where possible any compound with a pKa within physiological range should be considered
with extra care.
When considering the method for analysis, a wide range of techniques are available. Some
of the assay methods have been validated, but many are out dated and more accurate
options now available. Methods of analysis significantly affects the correlation and in
particular, compounds with limited absorbance extinction coefficients. In particular
LC-MS/MS displayed excellent correlation on a wide range of compounds with impressive
limits of detection.
With regards to the method used for the extraction of saliva there have been many collection
methods tried, and although spitting into a container seems to be the easiest method in
adults and children, this is not practical for infants. When considering infants, it is thought
that wiping the mouth with sterile gauze and rinsing with 5ml of sterile water and waiting for 5
minutes helps to reduce the risk of contamination [81] from either oral medication or
mother’s milk. Some research has shown that turning the infant onto their side to let the
saliva pool in the cheek was the easiest method of collection [81]. Other authors have
extracted the saliva from under the tongue and have collected it using various devices such
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as pipettes [77], mucous extractors [18], adapted pacifiers [64], suction devices [24] [65] and
more recently salivettes/salimetrics collection devices [70].
There are advantages and disadvantages associated with all of these collection devices;
Pipettes have limitations due to saliva viscosity, the adapted pacifier is not applicable to all
infants and suction devices are more troublesome than the other methods. The Salivette®
collection device has been specifically designed for the purpose and consists of a cotton
swab which is placed in the mouth until saturated, and is then placed into its container where
the fluid can be collected. The only disadvantage recorded for this collection method is that
some drugs may absorb onto the cotton swab [82]. Despite this, it remains suitable for most
drugs.
The type of collecting tube also needs to be taken into account when beginning a saliva
collection. Studies have shown that tubes containing serum separator gels can significantly
affect the determination of some drugs such as phenytoin [83, 84] or ribavirin [85].
Therefore, it is strongly recommended to evaluate the matrix effect of collecting tubes [3].
Once collected, the majority of the samples that we considered were frozen at -20°C before
analysis with no detrimental effect. One study [42] reported better correlation with fresh
samples compared with frozen, however there was insufficient evidence to conclude that this
is the case for the majority of compounds. Finally, the question of whether to stimulate saliva
production when collecting the samples also needs addressed prior to creating a reliable
reproducible method. From the compounds studied, stimulation had limited effect on those
drugs with excellent correlation. This is most likely due to the method of stimulation using
critic acid, and thereby lowering saliva pH further and not effecting % ionization of the weakly
acidic or neutral drugs. Stimulation displayed a varied effect overall on the compounds, with
no apparent correlation with chemical properties, and therefore should only be considered if
necessary.
In conclusion, there is significant potential for infantile saliva testing, and in particular for
neutral and weakly acidic compounds.
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References
[1] A. Dasgupta, Handbook of Drug Monitoring Methods, New Jersey: Humana Press
Inc, 2008.
[2] N. Buchanan, "Therapeutic Drug Monitoring," Indian J Pediatr, vol. 53, no. 2, pp. 149-
162, 1986.
[3] W. Zhao and E. Jacqz-Aigrain, Principles of Therapeutic Drug Monitoring. In
Pediatric Clinical Pharmacology., vol. 205, Heidelberg, Ed., Berlin: Springer, 2011, pp. 77-
90.
[4] G. L. Kearns, S. M. Sbdel-Rahman, S. W. Alander, D. L. Blowey, J. S. Leeder and R.
E. Kauffman, "Developmental pharmacology—drug disposition, action, and therapy in infants
and children," N Engl J Med, vol. 18, no. 349, pp. 1157-1167, Sept 2003.
[5] P. A. Routledge, "Pharmacokinetics in children," Journal of antimicrobial
chemotherapy, vol. 34, no. Suppl A, pp. 19-24, 1994.
[6] G. Koren and R. Parker, "Interpretation of excessive serum concentrations of digoxin
in children," Am J Cardiol , vol. 55, no. 9, pp. 1210-1214, Apr 1985.
[7] G. Koren, "Therapeutic drug monitoring principles in the neonate," Clin Chem, vol.
43, no. 1, pp. 222-227, Jan 1997.
[8] G. W. t Jong, A. G. Vulto, M. de Hoog, K. J. Schimmel, D. Tibboel and J. N. Van den
Anker, "Unapproved and Off-Label Use of Drugs in a Children's Hospital," N Engl J Med, vol.
343, no. 15, pp. 1125-1125, Oct 2000.
[9] S. Conroy, I. Choonara, P. Impicciatore, A. Mohn, H. Arnell, A. Rane and et al,
"Survey of unlicensed and off label drug use in paediatric wards in European countries,"
BMJ, vol. 320, no. 7277, pp. 79-82, Jan 2000.
[10] A. S. Gross "Best practice in therapeutica drug monitoring," Br J Clin Pharmacol, vol.
46, pp.95-99, 1998.
[11] S Pichini, I. Alteri, P. Zuccaro, R. Pacifici. "Drug monitoring in non conventional
biological fluids and matrices," Clin Pharmakinet, vol. 3, pp. 211-228, Nov 1996.
22
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
[12] D. B. Hawcutt, A. C. Rose, S. Fuerst-Recktenwald, T. Nunn and M. A. Turner, Points
to consider when planning the collection of blood or tissue samples in clinical trials of
investigational medicinal products in children, infants and neonates. In Guide to Paediatric
Drug Developmentnd Clinical Research, v. d. A. J. Rose K, Ed., Washington D.C: Karger
Publishers, 2010.
[13] P. M. Edelbroek, J. Van Der Hejjden and L. M. Stolk, "Dried blood spot methods in
therapeutic drug monitoring: methods, assays, and pitfalls," Ther Drug Monit, vol. 31, no. 3,
pp. 327-336, Jun 2009.
[14] W. Li and F. L. Tse, "Dried blood spot sampling in combination with LC‐MS/MS for
quantitative analysis of small molecules," Biomed Chromat, vol. 24, no. 1, pp. 49-65, Dec
2010.
[15] R. E. Choo and M. A. Huestis, "Oral Fluid as a diagnostic Tool," Clin Chem Lab Med,
vol. 42, no. 11, pp. 1273-1287, Nov 2004.
[16] P. N. Patsalos and D. J. Berry, "Therapeutic Drug Monitoring of Antiepileptic Drugs
by use of Saliva," Ther Drug Monit, vol. 35, no. 1, pp. 4-29, Feb 2013.
[17] R. Gorodischer, P. Burtin, P. Hwang, M. Levine and G. Koren, "Saliva versus blood
sampling for therapeutic drug monitoring in children: patient and parental preferences and an
economic analysis," Ther Drug Monit, vol. 16, no. 5, pp. 437-443, October 1994.
[18] R. Gorodischer and G. Koren, "Salivary Excretion of Drugs in Children: Theoretical
and Practical Issues in Therapeutic Drug Monitoring," Developmental Pharmacology and
Therapeutics, vol. 19, no. 4, pp. 161-177, Jan 1992.
[19] S. A. Malone, M. J. Eadie, R. S. Addison, A. W. E. Wright and R. G. Dickson,
"Monitoring salivary lamotrigine concentrations," Clin Neurosci, vol. 13, no. 9, pp. 902-907,
Nov 2006.
[20] T. Gordi, T. N. Hai and N. M. Hoai, "Use of Saliva and capillary blood samples as
substitutes for venous blood sampling in pharmacokinetic investigations of artemisinin," Eur
J Clin Pharmacol, vol. 56, no. 8, pp. 561-566, Nov 2000.
23
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
[21] D. H. Huffman, "Relationship between digoxin concentrations in serum and saliva,"
Clin Pharmacol Ther, vol. 17, no. 3, pp. 310-312, Mar 1975.
[22] I. Tsiropoulos, O. Kristensen and N. A. Klitgaard, "Saliva and Serum Concentration of
Lamotrigine in Patients with Epilepsy," Ther Drug Monit, vol. 22, no. 5, pp. 517-521, Oct
2000.
[23] J. J. McAuliffe, A. L. Sherwin, I. E. Leppik, S. A. Fayle and C. E. Pippenger, "Salivary
levels of anticonvulsants A practical approach to drug monitoring.," Neurology, vol. 27, no. 5,
p. 409, May 1977.
[24] N. N. Khanna, H. S. Bada and S. M. Somani, "Use of salivary concentrations in the
prediction of serum caffeine and theophylline concentrations in premature infants.," J
Pediatr, vol. 96, no. 3, pp. 494-499, March 1980.
[25] M. Berkovitch, T. Bistritzer, M. Aladjem, P. Burtin, T. Dagan, Z. Chen-Levi and et al,
"Clinical relevance of therapeutic drug monitoring of digoxin and gentamicin in the saliva of
children," Therapeutic drug monitoring, vol. 20, no. 3, pp. 253-256, Jun 1998.
[26] J. Autmizguine, P. Brian Smith, M. Sampson, P. Ovetchkine, M. Cohen-Wolkowiez
and K. M Watt, "Pharmacokinetic studies in infants using minimal-risk study designs,"
Current clinical pharmacology, vol. 9, no. 4, pp. 350-358, Nov 2014.
[27] B. Bailey, J. Klein and G. Koren, "Noninvasive methods for drug measurement in
pediatrics," Pediatr Clin North Am, vol. 44, no. 1, pp. 15-26, Feb 1997.
[28] O. Technologies, 2017. [Online]. Available:
http://ovidsp.uk.ovid.com/sp-3.26.1a/ovidweb.cgi. [Accessed 14th August 2017].
[29] J. W. Toback, P. Gal, N. V. Erkan, C. Roop and H. Robinson, "Usefulness of
Theophylline Saliva Levels in Neonates.," Ther Drug Monit, vol. 5, no. 2, pp. 185-189, Jun
1983.
[30] I. A. Siegel, H. Ben-Aryeh, D. Gozal, A. A. Colin, R. Szargel and D. Laufer,
"Comparison of Unbound and Total Serum Theophylline Concentrations with Those of
Stimulated and Unstimulated Saliva in Asthmatic Children.," Ther Drug Monitor, vol. 12, no.
12, pp. 460-464, Sept 1990.
24
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
[31] M. Culea, N. Palibroda, P. Chereches-Panta and M. Nanulescu, "Comparison of
isotopic dilution methods for determination of theophylline in the plasma and saliva of infants
and children," Chromatographia, vol. 53, no. S1, pp. 387-389, Jan 2001.
[32] P. Chereches-Panta, M. V. Nanukescu, M. Culea and N. Palibroda, "Reliability of
salivary theophylline in monitoring the treatment for apnoea of prematurity," J Perinatol, vol.
27, pp. 709-712, Aug 2007.
[33] J. V. Aranda, D. S. Sitar, W. D. Parsons, P. M. Loughnan and A. H. Neims,
"Pharmacokinetic aspects of theophylline in premature newborns.," N Engl J Med, vol. 295,
no. 8, pp. 413-416, Aug 1976.
[34] L. Hendeles, S. Burkey, L. Bighley and R. Richardson, "Unpredictability of
theophylline saliva measurements in chronic obstructive pulmonary disease," J Allergy Clin
Immunol, vol. 60, no. 6, pp. 335-338, Dec 1977.
[35] J. V. Aranda and T. Turmen, "Methylxanthines in apnea of prematurity.," Clinics in
Perinatology, vol. 6, no. 1, pp. 87-108, Mar 1979.
[36] G. Levy, E. F. Ellis and R. Koysooko, "Indirect plasma-theophylline monitoring in
asthmatic children by determination of theophylline concentration in saliva.," Pediatrics, vol.
53, no. 6, pp. 873-876, Jun 1974.
[37] J. R. Koup and B. A. Hart, "Relationship between plasma and whole blood
theophylline concentration in neonates," J Pediatr, vol. 94, no. 2, pp. 320-321, Feb 1979.
[38] R. E. Behrmann and F. C. Battaglia, "Protein binding of human fetal and maternal
plasmas to salicylate," J Appl Physiol , vol. 22, pp. 125-130, 1967.
[39] S. Wallace, "Altered plasma albumin in the newborn infant.," Br J Clin Pharmacol,
vol. 4, no. 1, pp. 82-85, Feb 1977.
[40] P. L. Morselli and V. Rovei, "Placental transfer of pethidine and norpethidine and
their pharmacokinetics in the newborn," Eur J Clin Pharmacol, vol. 18, no. 1, pp. 25-30, Jan
1980.
[41] C. E. Cook, E. Amerson, W. K. Poole, P. Lesser and L. O'Tuama, "Phenytoin and
Phenobarbital concentrations in saliva and plasma measured by radiommunoassay," Clin
25
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
Pharmacol Ther, vol. 18, no. 6, pp. 742-747, Dec 1975.
[42] R. F. Goldsmith and R. A. Ouvrier, "Salivary anticonvulsant levels in children: a
comparison of methods," Ther Drug Monit, vol. 3, no. 2, pp. 151-158, 1981.
[43] J. C. Mucklow, C. J. Bacon, A. M. Hierons, J. K. Webb and M. D. Rawlins,
"Monitoring of Phenobarbitone and Phenytoin Therapy in Small Children by Salivary
Samples," Ther Drug Monit, vol. 3, no. 3, pp. 275-277, Jan 1981.
[44] M. Lifshitz, Z. Ben-Zvi and R. Gorodischer, "Monitoring Phenytoin Therapy using
Citric Acid-Stimulated Saliva in infants and Children," Ther Drug Monit, vol. 12, no. 4, pp.
334-338, Feb 1990.
[45] T. Zysset, A. Rudeberg, F. Vassella, A. Kupfer and J. Bircher, "Phenytoin therapy for
epileptic children: evaluation of salivary and plasma concentrations and of methods of
assessing compliance.," Dev Med Child Neurol, vol. 23, no. 1, pp. 66-75, Feb 1981.
[46] A. S. Troupin and P. Friel, "Anticonvulsant level in saliva, serum, and cerebrospinal
fluid.," Epilepsia, vol. 16, no. 2, pp. 223-227, Jun 1975.
[47] M. G. Horning, L. Brown, J. Nowlin, K. Letratanangkoon, P. Kellaway and T. E. Zion,
"Use of saliva in therapeutic drug monitoring.," Clin Chem, vol. 23, no. 2, pp. 157-164, Feb
1977.
[48] D. Schmidt and H. J. Kupferberg, "Diphenylhydantoin, phenobarbital, and primidone
in saliva, plasma, and cerebrospinal fluid," Epilepsia, vol. 16, no. 5, pp. 735-741, Dec 1975.
[49] G. F. Blom and P. J. Guelen, The distribution of antiepileptic drugs between serum,
saliva and cerebrospinal fluid in: Antiepileptic Drug Monitoring, Pitman, Ed., London:
Pippenger, Eds, 1977.
[50] R. Gorodischer, P. Burtin, Z. Verjee, P. Hwang and G. Koren, "Is Saliva Suitable for
Therapeutic Monitoring of Anticonvulsants in Children: An Evaluation in the Routine Clinical
Setting," Ther Drug Monit, vol. 19, no. 6, pp. 637-642, Dec 1997.
[51] J. C. Mucklow, M. R. Bending, G. C. Kahn and C. T. Dollery, "Drug Concentration in
Saliva," Clini Pharmacol Ther, vol. 24, no. 5, pp. 563-570, Nov 1978.
26
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
[52] T. A. Moreland, D. A. Priestman and G. W. Rylance, "Saliva carbamazepine levels in
children before and during multiple dosing," Br J Clin Pharmacol, vol. 13, no. 5, pp. 647-651,
May 1982.
[53] C. Knott and F. Reynolds, "The place of saliva in antiepileptic drug monitoring," Ther
Drug Monit, vol. 6, no. 1, pp. 35-42, Mar 1984.
[54] I. M. Friedman, I. F. Litt, R. Henson, D. Holtzman and D. Halverson, "Saliva
phenobarbital and phenytoin concentrations in epileptic adolescents.," J Pediatr, vol. 98, no.
4, pp. 645-647, Apr 1981.
[55] K. Tokugawa, K. Ueda, H. Fujito and T. Kurokawa, "Correlation between the saliva
and free serum concentration of phenobarbital in epileptic children," Eu J Clin Pharmacol,
vol. 145, no. 5, pp. 401-402, Oct 1986.
[56] M. V. Miles, M. B. Tennison, R. S. Greenwood and et al, ". Evaluation of the Ames
Seralyzer for the determination of carbamazepine, phenobarbital, and phenytoin
concentrations in saliva.," Ther Drug Monit, vol. 12, no. 5, pp. 501-510, Sept 1990.
[57] M. V. Miles, M. B. Tennison and R. S. Greenwood, "Intraindividual variability of
carbamazepine, phenobarbital, and phenytoin concentrations in saliva.," Ther Drug Monit,
vol. 13, no. 2, pp. 166-171, Mar 1991.
[58] E. Zalzstein, N. Zucker and M. Lifshitz, "Digoxin concentration in saliva and plasma
in infants, children, and adolescents with heart disease," Current Therapeutic Research, vol.
64, no. 9, pp. 743-749, Nov 2003.
[59] N. Krivoy, N. Rogin, Z. Greif, H. Ben-Aryeh, D. Gutman and G. Alroy, "Relationship
between digoxin concentration in serum and saliva in infants," J Pediatr, vol. 99, no. 5, pp.
810-811, Nov 1981.
[60] M. Berkovitch, M. Goldman, R. Silverman, Z. Chen-Levi, R. Greenberg, O. Marcus
and et al, "Therapeutic drug monitoring of oce daily gentamycin in serum and saliva in
children," Eu J Pediatr, vol. 159, pp. 697-698, Aug 2000.
[61] S. Mahmod, M. H. Al-Hakiem, J. Landon, D. S. Smith and E. J. Shaw,
"Aminoglycoside antibiotics do not appear in saliva," Clin Chem, vol. 29, no. 5, pp. 988-989,
27
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
May 1983.
[62] E. A. Kopecky, S. Jacobson, J. Klein, B. Kapur and G. Koren, "Correlation of
Morphine Sulfate in Blood Plasma and Saliva in Pediatric Patients," Ther Drug Monit, vol.
19, no. 5, pp. 530-534, Jun 1997.
[63] M. Ryan, S. A. Grim, M. V. Miles, P. H. Tang, T. A. Fakhoury, R. H. Strawsbury and
et al, "Correlation of Lamotrigine Concentrations Between Serum and Saliva,"
Pharmacotherapy, vol. 23, no. 12, pp. 1550-1557, Dec 2003.
[64] M. Rauh, D. Stachel, M. Kuhlen, M. Groschl, W. Holter and W. Rascher,
"Quantification of busulfan in Saliva and Plasma in Haematopoietic Stem Cell
Transplantation in Children," Clin Pharmacokin, vol. 45, no. 3, pp. 305-316, Mar 2006.
[65] K. C. M. Van Der Elst, M. Van Alst, M. N. Lub-de Hooge, K. Van Hateren, J. G.
Kosterink, J. W. Alffennaar and et al, "Clinical Validation of the Analysis of Fluconazole in
Oral Fluid in Hospitalised Children," Antimicro agents Chemother, vol. 58, no. 11, pp. 6742-
6746, Nov 2014.
[66] S. N. De Wilt, K. T. M. Kerkvliet, M. G. A. Wezenberg, S. Ottink, W. C. Hop, A. G.
Vulto and et al, "Use of Saliva in Therapeutic Drug Monitoring of Caffeine in Preterm
Infants," Ther Drug Monit, vol. 23, no. 3, pp. 250-254, June 2001.
[67] H. S. Bada, N. N. Khanna, S. M. Somani and A. A. Tin, "Interconversion of
theophylline and caffeine in newborn infants," J Pediatr, vol. 94, no. 6, pp. 993-995, Jun
1979.
[68] T. C. Lee, B. G. Charles, P. A. Steer and V. J. Flenady, "Saliva as a Valid Alternative
to Serum in Monitoring Intravenous Caffeine Treatment for Apnea of Prematurity," Ther Drug
Monit, vol. 18, no. 3, pp. 288-293, Jun 1996.
[69] F. Z. Chioukh, A. Chaabane, H. B. Hamida, K. Ben Ameur, K. Aouam and K.
Monastiri, "Saliva as an alternative to plasma in therapeutic drug monitoring of caffeine in
preterm infants," J Matern Fetal Med, vol. 27, no. S1, p. 398, 2014.
[70] N. R. Dobson, X. Liu, L. M. Rhein, R. A. Darnall, M. J. Corwin, B. L. McEntire and et
al, "Salivary caffeine concentrations are comparable to plasma concentrations in preterm
28
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
772
773
infants receiving extended caffeine therapy," Br J Clin Pharmacol, vol. 82, pp. 754-761, Apr
2016.
[71] K. Y. Chee, D. Lee, D. Byron, D. Naidoo and A. Bye, "A simple collection method for
saliva in children: potential for home monitoring of carbamazepine therapy," Br J Clin
Pharmacol, vol. 35, no. 3, pp. 311-313, Mar 1993.
[72] H. Bartels, E. Gunther and S. Wallis, "Flow-dependent salivary primidone levels in
epileptic children," Epilepsia, vol. 20, no. 4, pp. 431-436, Aug 1979.
[73] W. Schramm, T. M. Annesley, G. J. Siegel, J. C. Sakellares and R. H. Smith,
"Measurement of Phenytoin and Carbamazepine in an Ultrafiltrate of Saliva.," Ther Drug
Monitor, vol. 13, no. 5, pp. 452-460, Sep 1991.
[74] H. G. Westenberg, E. Van Der Kleijn, T. T. Oei and R. A. De Zeeuw, "Kinetics of
carbamazepine and carbamazepine‐epoxide, determined by use of plasma and saliva," Clin
Pharmacol and Ther, vol. 23, no. 3, pp. 320-328, Mar 1978.
[75] R. Chambers, M. Homeida, K. R. Hunter and R. H. Teague, "Salivary carbamazepine
concentrations," The Lancet, vol. 309, no. 8012, pp. 656-657, Mar 1977.
[76] G. W. Rylance, T. A. Moreland and G. M. Butcher, "Carbamazepine dose-frequency
requirement in children," Arch Dis Child , vol. 54, no. 6, pp. 454-458, Jun 1979.
[77] L. Rui-Rui, X. Y. Sheng, L. Y. Ma, H. X. Yao, L. X. Cai, C. Y. Chen and et al, "Saliva
and Plasma Monohydroxcarbazepine Concentrations in Pediatric Patients With Epilepsy,"
Ther Drug Monit, vol. 38, no. 3, pp. 365-370, Jun 2016.
[78] B. B. Gallagher and I. P. Baumel, "Primidone. Absorption, distribution, and
excretion," Antiepileptic Drugs, pp. 357-359, 1972.
[79] M. Tennison, I. Ali and M. V. Miles, "Feasibility and Acceptance of Salivary
Monitoring of antiepileptic Drugs via the US Postal Service," Ther Drug Monit, vol. 26, no. 3,
pp. 295-299, June 2004.
[80] A. Tal, M. Aviram and R. Gorodischer, "Variations in theophylline concentrations
detected by 24 hour saliva concentration profiles in ambulatory children with asthma," J
Allergy Clin Immunol , vol. 86, no. 2, pp. 238-243, Aug 1990.
29
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
801
[81] H.P.Chang, G.C. Anderson and C.E.Wood. “Feasible and valid saliva collection for
cortisol in transitional newborn infants”. Nursing Research, vol. 44, no. 2, pp. 117-119, Mar
1995.
[82] A.M. Bermejo, A.C.S. Lucas and M.J. Tabernero. “Saliva/plasma ratio of methadone
and EDDP”. J Anal Toxicol, vol. 24, no. 1, pp. 70-72, Jan, 2000.
[83] F. Quattrocchi, H.T. Karnes, J.D. Robinson and L. Hendeles. “Effect of Serum Separator
Blood Collection Tubes on Drug Concentrations”. Ther Drug Monit. Vol. 5, no.3 , pp. 359-
362, Sep 1983.
[84] A. Dasgupta, R. Dean, S. Saldana, G. Kinnaman and R.W. McLawhon. “Absorption of
Therapeutic Drugs by Barrier Gels in Serum Separator Blood Collection Tubes: Volume-and
Time-dependent Reduction in Total and Free Drug Concentrationsa”. Am J Clin Pathol, vol.
101, no. 4, pp. 456-461, Jan, 1994.
[85] P. Marquet, F.L. Sauvage, V. Loustaud-Ratti, G. Babany, A. Rousseau and G. Lachatre.
“Stability of ribavirin concentrations depending on the type of blood collection tube and
preanalytical conditions”. Ther Drug Monit, vol. 32, no. 2, pp. 237-241, Apr, 2010.
[86] R. Koysooko, E.F. Ellis and G. Levy. “Relationship between theophylline concentration
in plasma and saliva of man”. Clin Pharmacol and Ther, vol. 15, no. 5, pp. 454-460, May,
1974.
[87] F. Plavsic, J. Culig, I. Bakran and B.Vrhovac. “Theophylline concentration in saliva as a
guide for individualization of its therapeutic use”. Brit J Clin Pharmaco, vol. 1, no 11, pp 533-
4, May 1981.
[88] J. Culig, A. Johnston and P. Turner, "Saliva theophylline concentrations after a single
oral dose.," Br J Clin Pharmacol, vol. 13, no. 2, pp. 243-245, 1982.
[89] C.H. Knott, M. Bateman and F. Reynolds. “Do saliva concentrations predict plasma
unbound theophylline concentrations? A problem re‐examined”. Brit J Clin Pharmaco, vol.1,
no. 17, pp9-14, Jan 1984.
[90] P. Ebden, D. Leopold, D. Buss, A.P. Smith and P.A. Routledge. “Relationship between
saliva and free and total plasma theophylline concentrations in patients with chronic airflow
30
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
828
829
obstruction”. Thorax, vol.1, no.40, pp. 526-529, Jul 1985.
[91] J.W. Paxton, B. Whiting and K.W. Stephen. “Phenytoin concentrations in mixed, parotid
and submandibular saliva and serum measured by radioimmunoassay”. Brit J Clin
Pharmaco, vol.1, no.4, pp.185-191, Apr 1977.
[92] C. Knott, A. Hamshaw-Thomas and F. Reynolds. “Phenytoin-valproate interaction:
importance of saliva monitoring in epilepsy”. Br Med J, vol.2, no. 284, pp.3-6, Jan 1982.
[93] L.M. Tsanaclis, J. Allen, E. Perucca et al. “Effect of valproate on free plasma phenytoin
concentrations”. Br J Clin Pharmacol, vol.18, no.1, pp. 17-20, Jul 1984.
[94] H.G. Van. “Comparative study of the levels of anticonvulsants and their free fractions in
venous blood, saliva and capillary blood in man”. J Pharmacol, vol.15, no.1, pp 27-35, Jan
1984.
[95] C. Knott, C.P. Williams and F. Reynolds. “Phenytoin kinetics during pregnancy and the
puerperium”. BJOG: An International Journal of Obstetrics & Gynaecology, vol. 1, no. 93, pp.
1030-1037, Oct 1986.
[96] P.V. Luoma, J.E. Heikkinen and P.R. Ylostalo. “Phenobarbital pharmacokinetics and
salivary and serum concentrations in pregnancy”. Ther Drug Monit, vol. 4, pp. 65-68, Mar
1982.
[97] D. Shen. “Saliva phenobarbital concentration in epileptics. Chung Hua Shen Ching
Ching Shen Ko Tsa Chih, vol. 22, no. 6, pp. 369-370, 1989.
[98] W.J. Jusko, L. Gerbracht, L.H. Golden and J.R. Koup, J. R. “Digoxin concentrations in
serum and saliva”. Commun Chem Pathol Pharmacol, vol.10, no.1, pp. 189-192, Jan 1975.
[99] W.J.F, Van der Vijgh. “Comparison of salivary digoxin concentration with plasma levels
in man”. Neth. J. Med, vol. 18, pp. 269-272, 1975.
[100] P.H. Jourbert, F.O. Muller and B.M. Aucamp. “Salivary digoxin concentration in saliva
and serum”. Brit J Clin Pharmaco, vol. 3, pp. 673-674, Aug 1976.
[101] H. Allonen, E. Iisalo, L. Kangas, R. Lammintausta and M. Salonen. “Estimation of
pharmacokinetic parameters of digoxin from serum, saliva and urine”. Int J Clin Pharmacol
Biopharm, vol. 16, no. 9, pp.420-423, Sept 1978.
31
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
[102] J. Lazowski, A. Lypka, and P. Borkowski. “The relationship between digoxin
concentration in saliva and serum”. Polski tygodnik lekarski, vol. 30, no. 33, pp. 1709 -1711,
Oct 1978.
[103] S. Mahmod, D.S. Smith and J. Landon. “Radioimmunoassay of salivary digoxin by
simple adaptation of a kit method for serum digoxin: saliva/serum ratio and correlation”. Ther
Drug Monit, vol. 1, no. 9, pp. 91-96, Mar 1987.
[104] A.F. Cohen, L. Ashby, D. Crowley et al. “Lamotrigine (BW430C), a potential
anticonvulsant. Effects on the central nervous system in comparison with phenytoin and
diazepam. Br J Clin Pharmacol, Vol. 20, no. 6, pp. 619-629, Dec 1985.
[105] Z. Trnavska, H. Krejcova, Z. Ykaczykovam et al. “Pharmacokinetics of lamotrigine
(Lamictal) in plasma and saliva”. Eur J Drug Metab Pharmacokinet, vol. 3, pp. 211-215, Jan
1991.
[106] T. Incecayir, I. Agabeyoglu and K.Gucuyener. “Comparison of plasma and saliva
concentrations of lamotrigine in healthy volunteers. Arzneimittelforschung, vol. 57, no. 8, pp.
517-521, Aug 2007.
[107] C.H. Koks CH, K.M. Crommentuyn, R.M. Hoetelmans, R.A. Mathôt RA and J.H.
Beijnen. “Can fluconazole concentrations in saliva be used for therapeutic drug monitoring?”
Ther Drug Monit, vol. 1, no.23, pp. 499-453, Aug 2001.
[108] C.H. Koks, H. Rosing, P.L. Meenhorst, A. Bult and J.H. Beijnen. “High-performance
liquid chromatographic determination of the antifungal drug fluconazole in plasma and saliva
of human immunodeficiency virus-infected patients”. J Chromatog, vol. 20, no. 663, pp. 345-
351, Jan 1995.
[109] J.W. Paxton and R.A. Donald. “Concentrations and kinetics of carbamazepine in whole
saliva, parotid saliva, serum ultrafiltrate, and serum”. Clin Pharmacol Ther, vol. 28, no. 5, pp.
695-702, Nov 1980.
[110] O. Kristensen and H.F. Larsen. “Value of saliva samples in monitoring carbamazepine
concentrations in epileptic patients”. Acta Neurol Scand, vol. 1, no. 61, pp. 344-350, Jun
1980.
32
858
859
860
861
862
863
864
865
866
867
868
869
870
871
872
873
874
875
876
877
878
879
880
881
882
883
884
885
[111] J.J. MacKichan, P.K. Duffner and M.E. Cohen. “Salivary concentrations and plasma
protein binding of carbamazepine and carbamazepine-10, 11-epoxide in epileptic patients”.
Br J Clin Pharmacol, vol. 12, no. 1, pp. 31-37, Jul 1981.
[112] A. Vasudev, K.D. Tripathi and V. Puri. “Correlation of serum and salivary
carbamazepine concentration in epileptic patients: implications for therapeutic drug
monitoring”. Neurology India, vol. 1, no. 50, pp. 60, Jan, 2002.
[113] S. Đorđević, V. Kilibarda and T. Stojanović. “Determination of carbamazepine in serum
and saliva samples by high performance liquid chromatography with ultraviolet detection”.
Vojnosanitetski pregled, vol. 66, no.5, pp. 347-352, 2009.
[114] S. Djordjević, V. Kilibarda, S. Vučinić, T. Stojanović and B. Antonijević. “Toxicokinetics
and correlation of carbamazepine salivary and serum concentrations in acute poisonings”.
Military Medical and Pharmaceutical Journal of Serbia, vol. 1, no. 69, pp. 389, May 2012.
[115] M. Thiesohn and G. Heimann. “Disposition of the anti-epileptic oxcarbazepine and its
metabolites in healthy volunteers”. Eur J Clin Pharmacol, vol. 1, no. 22, pp. 545-551, Aug
1982.
[116] O. Kristensen, N.A. Klitgaard, B. Jonsson et al. “Pharmacokinetics of 10-OH-
carbazepine, the main metabolite of the antiepileptic oxcarbazepine from serum and saliva
concentrations”. Acta Neurol Scand, vol. 1, no. 68, pp. 145-150, Sept 1983.
[117] N. A. Klitgaard and O. Kristensen, "Use of saliva for monitoring oxcarbazepine therapy
in epileptic patients.," Eu J Clin Pharmacol, vol. 31, no. 1, pp. 91-94, Jan 1986.
[118] J. M. Cardot, P. Degen, G. Flesch, P. Menge and W. Dieterle, "Comparison of plasma
and saliva concentrations of the active monohydroxy metabolite of oxcarbazepine in patients
at steady state," Biopharm Drug Dispos, vol. 16, no. 7, pp. 603-614, Oct 1995.
[119] M. V. Miles, P. H. Tang and M. A. Ryan, "Feasibility and limitations of oxcarbazepine
monitoring using salivary monohydroxycarbamazepine (MHD).," Therapeutic Drug
Monitoring, vol. 1, no. 26, pp. 300-304, June 2004.
[120] E. Zylber‐Katz, L. Granit and M. Levy. “Relationship between caffeine concentrations in
plasma and saliva”. Clin Pharmacol and Ther, vol. 1, no. 36, pp. 133-137, Jul 1984.
33
886
887
888
889
890
891
892
893
894
895
896
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
913