Keywords
dental local anesthesia - lidocaine - epinephrine - composition - pH - efficacy -
antibacterial activity and cytotoxicity
Introduction
Local anesthesia laid the fundamental foundation for pain regulation in dentistry.
The backbone of pain management was founded by William Halsted in 1885 by introducing
injectable dental local anesthesia, which reformed dental surgery.[1] Since the beginning of this revolution, a perceptible advancement in dental anesthesiology
has been apparent in the anesthetic solutions used.[2]
Despite multitudinous developments in medical and dental sciences, there are still
many local and systemic complications encountered in a clinical setting; pain at the
injection site, reduced efficacy, ulceration and induced infection at site of injection
are the few local complications. At the same time, toxicity is a major systemic complication
encountered.[3]
A practitioner preferably needs to have a requisite understanding concerning composition,
pH, efficacy, antibacterial activity and cytotoxicity of different anesthetic solutions
available to avoid these postoperative complications.
In recent years, different techniques have been proposed to mollify the intensity
of pain during the process of injecting an anesthetic. One of these is to use buffered
solutions of local anesthesia as recommended in a Cochrane study. Adding a buffer-like
bicarbonate with lidocaine increases the pH of the anesthetic solution, thus reducing
the pain during the injection. Pain is ascribed with local anesthetic’s acidity; hence,
practitioners should know about the pH and presence of bicarbonate ions in the anesthetic
solutions used in clinical practice.[3]
[4]
Attaining profound anesthesia is essential before starting any dental procedure. A
dental practitioner has a wide assortment of options in anesthetic solutions. The
difference in their efficacy is of paramount importance. Predominantly, all the solutions
available in the market are effective and safe, but still numerous failures of these
solutions are mentioned in literature and have been reported.[3]
[5]
Several factors play a role in increasing the efficacy of any local anesthetic agent.
The pH of local anesthetic solutions is an essential parameter to augment its efficacy.
Hogen et al demonstrated that the pH of commercially available local anesthetics in
combination with vasoconstrictors is approximately 4.5.[6] As the pH reduces, the local anesthetic efficacy is reduced (as in acidic conditions,
the number of ionized fractions dominates in the solution as compared with unionized
fractions), thus lowering the volume of local anesthesia accessible to obstruct the
sodium channels. Inflammatory conditions like abscess, pulpitis, and apical periodontitis
result in reducing the success of local anesthetics. In inflamed tissues, as a result
of the accumulation of lactic acid and its byproducts, the pH of the solution drops
to 0.5 to 1.0 resulting in acidosis. The pKa of commercially available local anesthetics is more than 7.5.[7]
[8]
[9] Therefore, in acidic conditions local anesthetics will have a diminished membrane
permeability, thereby having a reduced efficacy.[3]
[6]
Complication due to infection is sparse as we use disposable needles in our clinical
practice, but it still occurs in many instances. When a needle penetrates through
a contaminated tissue, infection augments into deeper tissues. Simultaneously, multiplex
bacterial species colonize the oral cavity, the needle injection causes perforation
of the mucous membrane, resulting in entry of these colonized bacteria into the tissues.
Under these circumstances, “suppurative local infections or odontogenic bacteremia”
may result after using these anesthetic solutions. Ideally, local anesthetic solutions
should have superior antimicrobial properties; thus, there is a need to examine the
antimicrobial activity of commercially available local anesthetics in the market and
conclude if antibiotic prophylaxis is still needed.[3]
[10]
When toxic concentrations of anesthetic solution diffuse from the blood into the central
nervous system, systemic complications develop such as toxicity. In vitro studies
signify toxic adverse effects of these drugs on different tissues such as fibroblast,
corneal endothelial cells, human leukocytes, and articular chondrocytes.[3]
[11]
[12]
[13]
[14] Lidocaine, an amide anesthetic, reported numerous adverse effects when used in combination
with different vasoconstrictors and preservatives. Epinephrine, a common vasoconstrictor
used in combination with various anesthetic agents is reported to cause complications
such as necrosis, ischemia, palpitations, or even dysrhythmias.[15]
[16]
Due to an increase in the prevalence of chemical differences in the similar composition
of anesthetic solutions and introduction of newer locally made solutions in the global
market by different manufacturers, practitioners should use solutions that are standardized
and strictly meet Food and Drug Administration (FDA) requirements. The prospect of
this in vitro and clinical study was to assess local anesthetic formulations from
three different countries and compare them in terms of composition, pH, efficacy,
antibacterial activity, and cytotoxicity.
Materials and Methods
Sampling
Three different commercially available local anesthetic solutions (2% lidocaine and
1:100,000 epinephrine) were obtained. These were Septodont (Saint-Maur-des, France,
Lot number 002277), Medicaine (Huons Co.Ltd Gyeonggi-do, Korean, Republic, Lot number
GA8127) and HD-Caine (Synchro Pharmaceuticals, Kot-Lakhpat, Lahore, Pakistan, Lot
number D-1903). These were labeled as S, M, and H, respectively. A total of 150 samples
of each solution were taken. These were further divided into subgroups, each containing
10 samples each for separate testing of composition, pH, efficacy, antibacterial activity,
and cytotoxicity.
The Setting of the Study
Ethical approval for this study was taken from the institutional review committee
of Islamic International Dental College (Ref. no. IIDC/IRC/2020/06/007). The study
was performed at Caraway Pharmaceuticals (Islamabad, Pakistan), Armed Forces Bone
Marrow Transplant center (Islamabad, Pakistan) and Islamic International Dental College
(Islamabad, Pakistan), from July to September 2020. The study was divided into two
stages: in vitro and clinical.
Compositional Analysis
The high-pressure liquid chromatography (HPLC) (Shimadzu, Japan 20AT) was used for
compositional analysis of the local anesthetic solutions. An ultraviolet-visible spectrophotometry
(UV-VIS) detector was used for the analysis of lidocaine and epinephrine. An Agilent
120 series HPLC system was utilized to conduct this HPLC-UV analysis. An autosampler,
column oven, binary pump, and a degasser (Agilent Technologies, Palo Alto, California,
United States) were incorporated in the system. C18 column (4.6 × 150 mm, 4 µm, YMC
Co. Ltd., Kyoto, Japan) was used, and the sample solution was injected into it. The
flow rate was 1.0 mL/min, while the temperature of the column was maintained to 30°C.
Chemstation software (Agilent Technologies, California, United States) was used to
operate this HPLC system. A standard solution was prepared to be used as a gold yardstick
against which the composition of S, M, and H were compared.
Preparation of Standard Solutions
Standard solution of lidocaine and epinephrine were prepared by dissolving aliquots
of these solutions with mobile phase to yield solutions with final concentrations
of 36 mg for lidocaine and 0.02 mg for epinephrine. The concentrations of these internal
standards are similar to the concentration of compounds in the cartridge.
Lidocaine Analysis
Lidocaine detection was performed using reversed-phase HPLC analysis equipped with
a C18 column (4.6 × 150 mm, 4 µm,). The temperature was kept constant at 25°C while
the pH was maintained to 5.8 by using NaOH. A combination of methanol and sodium dihydrogen
phosphate was incorporated for the mobile phase. The wavelength was adjusted to 230
nm for UV detection. The flow rate of the sample was kept at 1.0 mL/min and 10 µL
volume was used in the sample injection. The retention time for lidocaine was 9.97
minutes.[17]
Epinephrine Analysis
Epinephrine detection was performed by using reversed- phase HPLC analysis using a
C18 column (4.61 × 50 mm, 4 µm,). Temperature and pH were maintained at 25°C and 3.1,
respectively. For the mobile phase, a combination of water, methanol, and acetic acid
was used. The wavelength was adjusted to 230 nm for UV detection. The retention time
for epinephrine was 2.74 minutes.[18]
Calculation of Results
The area under the curve was used to determine the concentration of lidocaine and
epinephrine in the samples according to the formula given below.[19]
pH
Each group comprising 10 cartridges was used to measure the pH of the samples using
InoLab pH meter. In total, 3.6 mL of anesthetic solution was poured into a beaker
and the electrode of the meter was dipped into it and held for a few minutes.
Efficacy, the Clinical Arm of the Study
A double-blinded, quasi-clinical trial was performed to distinguish and compare the
pulpal anesthesia achieved after administering buccal infiltration in the mandibular
first molar.
To estimate a mean difference in electric pulp tester (EPT) scores of 13.0, with a
study power of 80% at a 5% significance level, a sample size of 11 participants per
group was calculated.[20] Since we had three groups, the sample size for our study was estimated to be 33.
Screening of the Patients
Patients diagnosed with irreversible pulpitis (n = 33) visiting the operative department for root canal treatment were selected. Informed
consent was signed by the participants before the trial. The procedure and any potential
risk or benefits were explained to the patients.
A detailed medical history was taken and vitals were recorded. The following inclusion
and exclusion criteria were determined:
Inclusion Criteria
-
Male and Female patients between the age range of 18 to 45 years
-
Active pain on visual analog scale (>54 mm) A range between 54 and 144 mm on a Heft-Parker
visual analog scale represents mild-to-moderate pain.[21]
[22]
-
The patient felt a sharp localized pain when a cold stimulus (ethyl chloride sprayed
on cotton) was applied for 15 seconds on the buccal surface of the tooth[23]
[24]
-
Periapical radiographs revealed the absence of periapical radiolucency
-
Vital coronal pulp
Exclusion Criteria
-
Patients with a previous history of allergic reactions to local anesthetics
-
Any pathology present near the tooth-like abscess
-
Presence of any systemic disease according to American Society of Anesthesiologists
(ASA) physical status classification[25]
-
Patients who took medication like relaxants or analgesics within 24 hours of injecting
the local anesthetic[26]
-
History of trauma and smoking (a potential confounder)
Investigational Phase
The label of the manufacturer was covered by an assistant to assure blinding so that
the dentist (one who administered the local anesthesia), and the patient were completely
unaware of the formulation that was being administered to avoid biases. Same volume
(1.8 mL) of local anesthetic solution was administered in the buccal fold of mandibular
first molar by the same dentist so there was no biasness in the injecting technique
either. A 30-gauge short needle was used, and the solution was injected steadily.
After 10 minutes, a cold test was performed on the patients. Patients with a negative
response to cold test were included in the study to ensure that buccal infiltration
was successful. Anesthetic efficacy was evaluated by using a commercially available
EPT (Denjoy). A rubber dam was placed on the tooth to be tested and was completely
dried with cotton wool, and then an acetate strip was placed between the contacts.
A small quantity of toothpaste was placed on the tip of the tester as a conducting
medium between the tooth and the tester. Next, the tip was placed in middle one-third
of the buccal surface. The electrode was placed on the lip to complete the circuit,
and the readings were taken 10 minutes after administrating the local anesthetic solution.
Patients were asked to respond by raising their hand when they felt any kind of tingling
sensation. Two consecutive values of 80 units showed that a profound pulpal anesthesia
was achieved.[26]
Antibacterial Activity
Tryptic soy agar (TSA) plates were used to culture and purify five different bacterial
strains as shown is [Table 1]. These cultures were attained from stock cultures of the biotechnology department
of Quaid-e-Azam University, Islamabad, Pakistan. A Negative Control (0.9% Sterile
Saline) (NaCl) (Grow-cells, United States) and a Positive Control (20 mL ampicillin
sodium salt; Gibco, Grand Island, New York, United States) were also acquired. Antibacterial
was investigated by using a broth dilution method. The bacterial strains were inoculated
in a tube of tryptic soy broth (TSB). The turbidity index was set to McFarland standard
10[5] organisms per mL by using a UV/VIS spectrophotometer (SAILAB, AY 1708008, AE-S90MD).
About 2 mL of all the three anesthetic formulations along with the control solutions
were poured into the test tubes and then added 1 mL of broth culture into each. The
calibration of the dilution loop was set to 0.01 mL and the samples were streaked
onto TSA agar plates. The plates were then placed in an incubator for 24 hours at
37°C. Next day, the number of bacterial colonies formed were counted manually.[27]
Table 1
Types of bacterial strains used in the study
|
Bacterial strains
|
|
Staphylococcus aureus (ATCC 6538)
|
|
Pseudomonas aeruginosa (ATCC 9721)
|
|
Klebsiella pneumoniae (ATCC 4619)
|
|
Bacillus subtilis (ATCC 6633)
|
|
Staphylococcus epidermidis (ATCC 1228)
|
Cytotoxicity Assay
Mesenchymal Stem Cells (MSCs) of a 24-year-old male were collected from the repository
of Air Force Bone Marrow Transplant Center (Islamabad, Pakistan). About 500 mL of
Dulbecco’s modified Eagle’s medium (DMEM), and 10% fetal bovine serum were used for
cell culturing along with 200 mL of heparin. CO2 incubator was utilized for cell expansion, which was done over a period of 5 days.
On the sixth day, the cultured cells were exposed to 0.25 mL sample solutions (group
S, M, and H) along with control solution (Dulbecco’s phosphate buffer saline Solution
[DPBS]; Gibco, Denmark) for 1 hour, after which the nonadherent cells were centrifuged
to remove the nonadherent cells. For complete removal of the local anesthesia, the
cells were cleaned with DPBS and were again cultured and kept for 24 hours. Next day,
7-aminoactinomycin D (AAD) stain was utilized to determine cell viability. The centrifuging
machine was set at 660 G/8 min, and Eppendorf tubes with cell cultures were centrifuged.
Next, cell cultures along with 2 µL of 7-AAD stain were added in fluorescence-activated
cell sorting tube and flow cytometry was done by using a (Navios; Beckman Counter).[27]
Statistical Analysis
Data was analyzed by using the Statistical Package for Social Science (SPSS for Windows
version 12, SPSS Inc., Chicago, Illinois, United States). Mean values (±standard error)
for various study attributes, that is, compositional analysis, pH, EPT scores, antibacterial
activity, and cytotoxicity were calculated. The magnitude of variation between three
groups, that is, S, M, and H was ascertained through ANOVA, using completely randomized
design (Snedecor and Cochran 1989). Duncan’s multiple range test was implied as a
post hoc test to detect differences between mean values (Duncan 1955). A p-value of 0.05 was arbitrarily considered to be significant.
Results
Compositional Analysis
HPLC analysis for the comparative evaluation of composition revealed peaks of standard
and sample solutions. [Table 2] gives description of peaks obtained from the curve of HPLC when the standard and
sample solutions were tested.
Table 2
High-pressure liquid chromatography results for standard and sample solutions (S,
M, and H)
|
Results
|
Lidocaine
|
Epinephrine
|
|
Standard
|
Septodont
|
Medicaine
|
HD-Caine
|
Standard
|
Septodont
|
Medicaine
|
HD-Caine
|
|
Retention time/min
|
9.97
|
9.97
|
9.97
|
9.97
|
2.74
|
2.74
|
2.74
|
2.74
|
|
Area of curve
|
8185478
|
8392210
|
8295329
|
8199829
|
24981
|
25987
|
31889
|
31054
|
|
Height of curve
|
262731
|
278431
|
262731
|
262731
|
184317
|
2659
|
194427
|
2459
|
|
Wavelength/nm
|
230 nm
|
230 nm
|
230 nm
|
230 nm
|
230 nm
|
230 nm
|
230 nm
|
230 nm
|
There was no difference in the lidocaine and epinephrine concentration between the
control solution and any of the local anesthetic solutions (p > 0.05). Also, no difference was found in the lidocaine and epinephrine concentration
between any of the three solutions S, M, or H (p > 0.05), as illustrated in [Tables 3] and [4] .
Table 3
Intergroup mean difference values and standard deviation (p values) for compositional
analysis, pH, efficacy, antibacterial activity and cytotoxicity
|
Agent
|
Comparative agent
|
Mean difference in lidocaine concentration (p-Value)
|
Mean difference in epinephrine concentration (p-Value)
|
Mean difference in pH (p-Value)
|
Mean difference in efficacy (p-Value)
|
Mean difference in Staphylococcus aureus (p-Value)
|
Mean difference in Klebsiella pneumonia (p-Value)
|
Mean difference in Bacillus subtilis (p-Value)
|
Mean difference in Staphylococcus epidermidis (p-Value)
|
Mean difference in Pseudomonas aeruginosa (p-Value)
|
Mean difference in cytotoxicity (p-Value)
|
|
Standard solution
|
Septodont
|
0.14 ± 0.07 (0.21)
|
0.001 ± 0.003 (0.99)
|
|
|
|
|
|
|
|
|
|
Medicaine
|
0.13 ± 0.07 (0.26)
|
0.002 ± 0.003 (0.94)
|
|
|
|
|
|
|
|
|
|
HD-Caine
|
0.02 ± 0.07 (0.99)
|
0.003 ± 0.003 (0.83)
|
|
|
|
|
|
|
|
|
|
Septodont
|
Medicaine
|
0.01 ± 0.06 (0.99)
|
0.003 ± 0.003 (0.83)
|
2.27 ± 0.004 (<0.01)
|
9.40 ± 0.89 (<0.01)
|
13.6 ± 7.0 (0.3)
|
5.3 ± 0.6 (0.0)
|
3.5 ± 1.2 (0.0)
|
110.9 ± 10.0 (0.0)
|
36.9 ± 2.7 (0.0)
|
0.1 ± 0.1 (0.6)
|
|
HD-Caine
|
0.12 ± 0.07 (0.33)
|
0.004 ± 0.003 (0.67)
|
0.57 ± 0.004 (<0.01)
|
1.20 ± 0.89 (0.38)
|
117.2 ± 7.0 (0.0)
|
10.1 ± 0.6 (0.0)
|
24.4 ± 1.2 (0.0)
|
137.8 ± 10.0 (0.0)
|
43.7 ± 2.7 (0.0)
|
0.6 ± 0.1 (0.0)
|
|
Medicaine
|
HD-Caine
|
0.11 ± 0.07 (0.41)
|
0.001 ± 0.003 (0.99)
|
1.70 ± 0.004 (<0.01)
|
8.20 ± 0.89 (<0.01)
|
103.6 ± 7.0 (0.0)
|
4.8 ± 0.6 (0.0)
|
20.9 ± 1.2 (0.0)
|
26.9 ± 10.0 (0.0)
|
6.8 ± 2.7 (0.1)
|
0.8 ± 0.1 (0.0)
|
|
Positive control
|
Septodont
|
|
|
|
|
209.5 ± 7.0 (0.0)
|
16.9 ± 0.6 (0.0)
|
39.9 ± 1.2 (0.0)
|
240.3 ± 10.0 (0.0)
|
61.4 ± 2.7 (0.0)
|
2.0 ± 0.1 (0.0)
|
|
Medicaine
|
|
|
|
|
195.9 ± 7.0 (0.0)
|
11.6 ± 0.6 (0.0)
|
36.4 ± 1.2 (0.0)
|
129.4 ± 10.0 (0.0)
|
24.5 ± 2.7 (0.0)
|
1.9 ± 0.1 (0.0)
|
|
HD-Caine
|
|
|
|
|
92.3 ± 7.0 (0.0)
|
6.8 ± 0.6 (0.0)
|
15.5 ± 1.2 (0.0)
|
102.2 ± 10.0 (0.0)
|
17.7 ± 2.7 (0.0)
|
2.7 ± 0.1 (0.0)
|
|
Negative control
|
|
|
|
|
272.5 ± 7.0 (0.0)
|
57.0 ± 0.6 (0.0)
|
121.1 ± 1.2 (0.0)
|
193.6 ± 10.0 (0.0)
|
60.9 ± 2.7 (0.0)
|
|
|
Negative control
|
Septodont
|
|
|
|
|
63.0 ± 7.0 (0.0)
|
40.1 ± 0.7 (0.0)
|
81.2 ± 1.2 (0.0)
|
46.7 ± 10.0 (0.0)
|
0.50 ± 2.7 (1.0)
|
|
|
Medicaine
|
|
|
|
|
76.6 ± 7.0 (0.0)
|
45.4 ± 0.7 (0.0)
|
84.7 ± 1.2 (0.0)
|
64.2 ± 10.0 (0.0)
|
36.4 ± 2.7 (0.0)
|
|
|
HD-Caine
|
|
|
|
|
180.2 ± 7.0 (0.0)
|
50.2 ± 0.7 (0.0)
|
105.6 ± 1.2 (0.0)
|
91.1 ± 10.0 (0.0)
|
43.2 ± 2.7 (0.0)
|
|
Table 4
Mean values for compositional analysis, pH, efficacy, antibacterial activity, and
cytotoxicity
|
Septodont
|
Medicaine
|
HD-Caine
|
Standard solution
|
Positive control
|
Negative control
|
|
Lidocaine concentration
|
36.14 ± 0.08
|
36.13 ± 0.04
|
36.02 ± 0.01
|
36.00 ± 0.00
|
|
|
|
Epinephrine concentration
|
0.021 ± 0.00
|
0.018 ± 0.00
|
0.017 ± 0.00
|
0.020 ± 0.00
|
|
|
|
pH
|
5.3 ± 0.002a
|
3.0 ± 0.002b
|
4.7 ± 0.003c
|
|
|
|
|
Efficacy
|
80.0 ± 0.0a
|
70.6 ± 0.9b
|
78.8 ± 0.4a
|
|
|
|
|
Staphylococcus aureus
|
209.5 ± 31.0a
|
195.9 ± 1.7a
|
92.3 ± 3.2b
|
|
0c
|
272.5 ± 10.3d
|
|
Klebsiella pneumonia
|
16.9 ± 0.5a
|
11.6 ± 0.6b
|
6.8 ± 0.2c
|
|
0d
|
57.0 ± 0.0e
|
|
Bacillus subtilis
|
39.9 ± 0.9a
|
36.4 ± 1.2a
|
15.5 ± 0.5b
|
|
0c
|
121.1 ± 1.2d
|
|
Staphylococcus epidermidis
|
240.3 ± 15.5a
|
129.4 ± 0.9b
|
102.5 ± 2.0b
|
|
0c
|
193.6 ± 2.5d
|
|
Pseudomonas aeruginosa
|
61.4 ± 3.0a
|
24.5 ± 1.5b
|
17.7 ± 1.5b
|
|
0c
|
60.9 ± 2.0a
|
|
Cytotoxicity
|
94.5 ± 0.1a
|
94.7 ± 0.0a
|
93.9 ± 0.0b
|
|
96.6 ± 0.0c
|
|
pH
The pH of S (5.30 ± 0.002) was found to be significantly greater than the pH of both
M (3.00 ± 0.002, p < 0.01) and H (4.7 ± 0.003, p < 0.05). H also had a significantly higher pH than M (p < 0.01; [Tables 3] and [4] ).
Efficacy
The results of efficacy obtained by EPT before and 10 minutes after administration
of local anesthetic solutions indicated that there was no difference in the mean EPT
values between Septodont (25.7 ± 0.7), Medicaine (25.6 ± 1.0) and HD-Caine (25.6 ±
0.8) recorded before the injection of the administration (p < 0.05). The EPT values 10 minutes after administering the injection were different
([Table 3]). S was found to have the highest efficacy (80.00 ± 0.00). This was followed by
H with an efficacy of 78.80 ± 0.40. However, the difference in efficacy between S
and H was not significant (p = 0.38). The efficacy of M (70.60 + 0.90) was significantly lesser than both S (p < 0.01) and H (p < 0.01).
Antibacterial Activity
The highest number of bacterial colonies were formed when bacterial cultures were
exposed to group S followed by group M and the least by Group H as illustrated in
[Fig. 1]. Therefore, demonstrating the highest antibacterial potential by group H and the
least by group S. The details are given in [Tables 3] and [4].[27]
Fig. 1 Comparative mean (±standard deviation) values of the number of bacterial colonies
for five bacterial species tested for group S, M, and H.
Cytotoxicity
The highest percentage viability was observed in the control group followed by group
S and M, while group H revealed the lowest viability of MSCs as illustrated in [Fig. 2]. A nonsignificant difference was observed when the MSCs were exposed to Septodont
and Medicaine, while the difference was significant when exposed to HD-Caine and control
solution, [Tables 3] and [4] gives the details of viability percentages along with their p-values.[27]
Fig. 2 Viability of mesenchymal monolayer cells for group S, M, and H as compared with control.
Discussion
This contradistinction study of different brands of anesthetic formulations was conducted
by analyzing the variance in their composition, pH, efficacy, antimicrobial properties,
and cytotoxicity, thus justifying their safe use. The study was conducted independently
without any form of financial or otherwise assistance from any of the product manufacturers,
hence free from any form of bias.
Several investigations seen in literature have adduced the significance of using HPLC
for the quantitative determination of amide linked local anesthetics and catecholamines
in pharmaceutical preparations.[18]
[28]
[29] Our study used HPLC to analyze the differences between the compositions of the three
dental anesthetics (Septodont, Medicaine, and HD-Caine) when compared with the standard
solutions. The outcome was evaluated by comparing the area under the curve of the
samples with the standard solutions. The concentrations of the lidocaine and epinephrine
standard solutions were 36 mg (2%) and 0.02 mg (0.001% or 1:100,000) respectively.
Our results suggested that there was no difference between the control solution’s
concentrations and any of the three solutions (S, M, and H). These results were in
line with the manufacturers’ claims.
A similar study was conducted by Salman et al in 2017. They analyzed and compared
the concentration of lidocaine HCL by HPLC using a UV detector at a wavelength of
254 nm at 25°C with a retention time of 19 minutes. They concluded that this methodology
offered easy and reliable results while utilizing a modest budget.[28] In another study, Mishra et al determined the concentration of epinephrine by HPLC.
They conducted the investigation using UV detector at a wavelength of 280 nm with
a retention time of 3.32 and 3.76 minutes. The results of his experiment revealed
the concentration of epinephrine to be 0.988 mg/mL while the manufacturer claim was
1 mg/mL. This novel finding suggested that this methodology could be routinely performed
for the determination of epinephrine in injections.[18]
The results of our study in terms of the pH of commercially available dental anesthetic
solutions are consistent with previous findings. All of the intergroup differences
in pH were found to be significant (p < 0.01). However, the range of these findings fell between the values of previous
findings by Hogan, Malamad, Mark, and Jason, which state that the normal physiological
pH of commercially available local anesthetics with vasoconstrictors is between 3
to 5.[6]
[30] To increase the shelf-life and escalate stability of anesthetic solutions, pharmaceutical
companies ensure that the solution’s pH is acidic. At a physiological pH of 7.4, adrenaline
is unstable and thus requires an acidic environment to remain stable. Moreover, in
an alkaline medium, lipid-soluble molecules diffuse out due to the low water solubility.[8] Acidic, water-soluble ionized molecules are present in dental cartridges, and these
molecules need to dissociate hydrogen ions in-order to become unionized. After this
conversion, the anesthetic solution can infiltrate the nerve sheath and block the
sodium gated channels, thereby preventing the generation of an action potential. Furthermore,
Shyamala et al stated that using agents with high pH can increase the anesthetic efficacy
as less time is taken by the body for unionized to ionized conversion.[6] The aromatic ring in the structure of a local anesthetic determines their potency
which depends on their lipid solubility. Lipid solubility increases the rate of diffusion
through cell membrane, so the proportion of anesthetic solution which is in lipid
soluble state determine their onset. Hydrochloric acid is added in the dental cartridges
to stabilize the solution, thus making molecules of anesthetic in a water-soluble
state while administering the injection. These water-soluble molecules need to be
converted to a lipid-soluble structure in order to facilitate diffusion into tissues
having a physiologic pH of 7.4 which is considerably lower than the pKa of anesthetics resulting in delayed onset of action. This explains the fact that
why there are more chances of local anesthetic failure in infectious cases like abscess
as these pathologies create an acidic environment resulting in metabolic acidosis,
thus lowering the pH of the tissues even below 7.4.[7]
[31]
Painless anesthesia is a major challenge for a dentist as this may cause an unpleasant
incident for a patient therefore discouraging them from seeking further treatments
especially pediatric dental patients. Consequentially, such patients prefer going
for general anesthesia either through IV or gaseous induction to reduce the distress
associated with the treatment.[32] Moreover, various behavior management techniques are also considered.[33] Ghazal et al conducted a randomized control trial and emphasized on the value and
use of visual information during dental procedure.[34] Studies have proved that pain while injecting the anesthetic is due to the acidity
of anesthetic solution along with other factors and thus various clinical studies
are focusing on the use of sodium bicarbonate in the solution to raise the pH more
than the physiological pH of the body. This would also make the anesthetic agent more
lipid-soluble.[35]
Local anesthetic efficacy is a multifarious matter, attained by several factors. The
study design was plotted to compare the anesthetic efficacy of the three brands of
anesthetic solutions commonly used in local market. The results observed in our study
were found in accordance with previous studies. The EPT values for all the three groups
increased with time after administrating the local anesthetic injection.[26] The EPT values for M recorded 10 minutes after the injection was significantly lower
than the values recorded for both S and H.
A similar study was performed by Abdul-Wahab and Boynes, which reported that the mean
increase in EPT reading for 2% lidocaine with 1:100,000 epinephrine was 43.5%. Participants
achieved profound pulpal anesthesia in 8 minutes when compared with other formulations
(but with different anesthetic agents). They further evaluated soft-tissue numbness,
which was achieved between 7 and 15 minutes for all the formulations.[26] Our study reported a mean increase of 54.3% for Group S, 45% for Group M, while
Group H showed a mean increase by 53.2% after 10 minutes. Another study by Meechan
et al compared the difference in efficacy achieved on mandibular first molar by buccal
infiltration and then with a combination of buccal and lingual infiltration using
2% lidocaine with 1:100,000 epinephrine. Their results showed that the same anesthetic
effect was produced either by using buccal infiltration alone or in combination with
lingual infiltration in mandibular first molar. Moreover, it took 10 to 14 minutes
to achieve peak anesthetic effect when EPT readings were recorded.[36] Literature reports that to attain an efficacious pulpal anesthesia in cases of irreversible
pulpitis, an inferior alveolar nerve block is not always an effective technique; therefore,
different techniques should also be considered.[37]
In a venture to analyze and determine a supercilious and suitable local anesthetic
brand, this study might help the clinicians to know the relative efficacy and what
to expect. In the case of clinical implication, using different local anesthetic agents
such as articaine may give superior results.[38] In this study, since the composition and concentration of cartridges was same, any
differences observed in efficacy might have due to the slight differences reported
for pH. A trend correlating pH with efficacy was observed in the study. As the acidity
decreased, the efficacy also decreased as shown by group M.
Antibacterial activity of anesthetics is a governing factor to inhibit infections
and therefore can serve as prophylaxis before a surgical procedure. Bacteremia, a
condition caused by the entry of numerous oral-microflora into the bloodstream, develops
when a local anesthetic injection damages the tissue at the point where its injected.[39] Postoperative dental procedures may supervene infections and injection abscesses,
refraining a patient’s healing process. Although these complications are infrequent,
they do transpire at various instances. They might emanate as a consequence of primary
infection when blood circulates from that site and when a contaminated needle penetrates
into deeper tissues or it might also be due to negligence of the dentist.[3]
[40] The quest of investigating the antibacterial activity of the three pharmaceutical
solutions was done to evaluate the most supercilious brand of anesthetic solution
that can be used to treat infections or as an irrigant. The findings of our study
knot well with the findings of Kesici et al and Aydin et al, the former demonstrated
a synergistic antibacterial effect of lidocaine, when used in combination with epinephrine[41] while the latter reported highest antibacterial activity of lidocaine in contrast
to other anesthetic agents.[42]
Our pharmaceutical industry has evolved over recent years. Drugs have different cytotoxic
receptors; some destroy the cell membrane or irreversibly bind with receptors while
others inhibit protein synthesis. Investigation of cell death by these damages necessitate
the use of cell viability assays which are established on diverse cell functions.[43] Cytotoxic analysis for the three groups revealed a statistically nonsignificant
difference between group S and M. While in comparison to these two groups, group H
and control revealed a significant difference. The findings of our study corroborated
with the findings of Wu et al, who concluded negligible toxic effects of lidocaine
in contrast to other anesthetic agents.[44] Shoshani et al deduced no percentage decrease in adipocyte viability when the cells
were exposed to different local anesthetic agents.[45] Clinical implications of a toxic anesthetic agent increase the likelihood of getting
oral paresthesia,[46]
[47] muscular stiffness,[48] and palatal ulceration.[49]
Limitations
This study had its limitations as well. Confounding factors such as any underlying
pathologies, operator technique or speed of injecting the solution and the fact affirming
the difficulties in achieving pulpal anesthesia in patients with pulpal pathologies
were not studied. Dentists need to apprehend these findings and then utilize them
in dental practice.
Conclusion
The composition of lidocaine and epinephrine were same as claimed by the manufacturers.
The pH of the three groups was significantly different; however, the range fell within
the pH of what the manufactures claim. The efficacies of Septodont and HD-Caine anesthetic
formulations were ideal.
Investigation of the stability of anesthetic solutions after forced degradation/aging
and to check for degradation products using HPLC is highly recommended for future
research. Furthermore, the evaluation of change in pH by the addition of bicarbonate
ions, and the possible correlation with reduction in pain during injection would have
been an advantage.
