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: b.callan@ulster.ac.uk

Marlene Sinclair

School of Nursing

Institute of Nursing and Health Research

Ulster University

Newtownabbey

Co Antrim

BT37 OQB

Telephone: 0044 28 9036 8118

Email:m.sinclair1@ulster.ac.uk

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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

[121] R. Newton, L.J. Broughton, M.J. Lind, P.J. Morrison, H.J. Rogers and I.D. Bradbrook.

“Plasma and salivary pharmacokinetics of caffeine in man”. Eu J Clin Pharmaco, vol. 1, no.

21, pp. 45-52, Jan 1981.

34

914

915

916