Oxylipins in Aqueous Humor of Primary Open-Angle Glaucoma Patients.

PURPOSE: Investigate the oxylipin profiles in the aqueous humor of primary open-angle glaucoma (POAG) patients. METHODS: Aqueous humor samples were collected from 17 POAG patients and 15 cataract subjects and subjected to a liquid chromatography/mass spectrometry (LC-MS) analysis to detect the oxylipins. The prediction potential of the differential abundant oxylipins was assessed by the receiver operating characteristic (ROC) curves. Pathway and correlation analyses on the oxylipins and clinical and biochemical parameters were also conducted. RESULTS: The LC-MS analysis detected a total of 76 oxylipins, of which 29 oxylipins reached the detection limit. The multivariate analysis identified five differential abundant oxylipins, 15-keto-prostaglandin F2 alpha (15-kPGF2α), Leukotriene B4 (LTB4), 12,13-Epoxyoctadecenoic acid (12,13-Epome), 15-Hydroxyeicosatetraenoic acid (15-HETE) and 11-Hydroxyeicosatetraenoic acid (11-HETE). The five oxylipins are enriched in the arachidonic acid metabolism and linoleic acid metabolism pathways. Pearson correlation analysis showed that 11-HETE was positively correlated with intraocular pressure and central corneal thickness and negatively with cup/disk area ratio in the POAG patients. In addition, 15-kPGF2α was moderately and positively correlated with the mean deviation (MD) of visual field defect, and LTB4 was moderately and negatively correlated with macular thickness. CONCLUSIONS: This study revealed the oxylipin profile in the aqueous humor of POAG patients. Oxylipins involved in the arachidonic acid metabolism pathway could play a role in POAG, and anti-inflammatory therapies could be potential treatment strategies for POAG.

Protein profile of purified plasma- and aqueous humor-derived extracellular vesicles from patients with ocular toxoplasmosis.

PURPOSE: To characterize the extracellular vesicle protein cargo in the aqueous humor and plasma of patients with ocular toxoplasmosis. METHODS: Aqueous humor and plasma were collected from six patients with active ocular toxoplasmosis and six patients with cataract. Extracellular vesicles were isolated, and western blotting and mass spectrometry were performed for protein analysis. RESULTS: All plasma samples from patients with ocular toxoplasmosis and cataract were positive for the tetraspanins CD63 and TSG101. However, the aqueous humor from patients with ocular toxoplasmosis was positive only for CD63. Sixty-seven new unreported proteins were identified in the aqueous humor and plasma of patients with the ocular toxoplasmosis and cataract. Of the 67 proteins, 10 and 7 were found only in the cataract and ocular toxoplasmosis groups, respectively. In general, these proteins were involved in immune system activation and retina homeostasis and were related to infections and retina-associated diseases. CONCLUSION: The distinct protein signatures between ocular toxoplasmosis and cataract may be helpful in the differential diagnosis of ocular toxoplasmosis. However, more studies are needed to better understand the role of these proteins in the pathogenesis of ocular toxoplasmosis.

Neurodegenerative biomarkers in different chambers of the eye relative to plasma: an agreement validation study.

BACKGROUND: Protein biomarkers have been broadly investigated in cerebrospinal fluid and blood for the detection of neurodegenerative diseases, yet a clinically useful diagnostic test to detect early, pre-symptomatic Alzheimer's disease (AD) remains elusive. We conducted this study to quantify Aß40, Aß42, total Tau (t-Tau), hyperphosphorylated Tau (ptau181), glial fibrillary acidic protein (GFAP) and neurofilament light chain (NfL) in eye fluids relative to blood. METHODS: In this cross-sectional study we collected vitreous humor, aqueous humor, tear fluid and plasma in patients undergoing surgery for eye disease. All six biomarkers were quantitatively measured by digital immunoassay. Spearman and Bland-Altman correlation analyses were performed to assess the agreement of levels between ocular fluids and plasma. RESULTS: Seventy-nine adults underwent pars-plana vitrectomy in at least one eye. Of the 79, there were 77 vitreous, 67 blood, 56 tear fluid, and 51 aqueous samples. All six biomarkers were quantified in each bio-sample, except GFAP and NfL in tear fluid due to low sample volume. All six biomarkers were elevated in vitreous humor compared to plasma samples. T-Tau, ptau181, GFAP and NfL were higher in aqueous than in plasma, and t-Tau and ptau181 concentrations were higher in tear fluid than in plasma. Significant correlations were found between Aß40 in plasma and tears (r = 0.5; p = 0.019), t-Tau in plasma and vitreous (r = 0.4; p = 0.004), NfL in plasma and vitreous (r = 0.3; p = 0.006) and plasma and aqueous (r = 0.5; p = 0.004). No significant associations were found for Aß42, ptau181 and GFAP among ocular fluids relative to plasma. Bland-Altman analysis showed aqueous humor had the closest agreement to plasma across all biomarkers. Biomarker levels in ocular fluids revealed statistically significant associations between vitreous and aqueous for t-Tau (r = 0.5; p = 0.001), GFAP (r = 0.6; p < 0.001) and NfL (r = 0.7; p < 0.001). CONCLUSION: AD biomarkers are detectable in greater quantities in eye fluids than in plasma and show correlations with levels in plasma. Future studies are needed to assess the utility of ocular fluid biomarkers as diagnostic and prognostic markers for AD, especially in those at risk with eye disease.

Proteomic Analysis of Aqueous Humor in Central Retinal Artery Occlusion: Unveiling Novel Insights Into Disease Pathophysiology.

Purpose: Central retinal artery occlusion (CRAO) is an ocular emergency that results from acute blockage of the blood supply to the retina and leads to a sudden vision loss. Other forms of ischemic retinopathies include diabetic retinopathy (DR), which involves chronic retinal ischemia and remains the leading cause of blindness in working-age adults. This study is the first to conduct a proteomic analysis of aqueous humor (AH) from patients with CRAO with a comparative analysis using vitreous humor (VH) samples from patients with DR. Methods: AH samples were collected from 10 patients with CRAO undergoing paracentesis and 10 controls undergoing cataract surgery. VH samples were collected from 10 patients with DR and 10 non-diabetic controls undergoing pars plana vitrectomy (PPV). Samples were analyzed using mass spectrometry. Results: Compared with controls, AH levels of 36 differentially expressed proteins (DEPs) were identified in patients with CRAO. Qiagen Ingenuity Pathway Analysis (IPA) revealed 11 proteins linked to ophthalmic diseases. Notably, enolase 2, a glycolysis enzyme isoform primarily expressed in neurons, was upregulated, suggesting neuronal injury and enzyme release. Additionally, clusterin, a protective glycoprotein, was downregulated. ELISA was conducted to confirm proteomics data. VH samples from patients with DR exhibited changes in a distinct set of proteins, including ones previously reported in the literature. Conclusions: The study provides novel insights into CRAO pathophysiology with multiple hits identified. Proteomic results differed between DR and CRAO studies, likely due to the different pathophysiology and disease duration. Translational Relevance: This is the first proteomic analysis of CRAO AH, with the potential to identify future therapeutic targets.

Green and sensitive detection of olopatadine in aqueous humor using a signal-on fluorimetric approach: GREEnness assessment.

Olopatadine (OLP) is widely utilized as an effective antihistaminic drug for alleviating ocular itching associated with allergic conjunctivitis. With its frequent usage in pharmacies, there arises a pressing need for a cost-effective, easily implementable, environmentally sustainable detection method with high sensitivity. This study presents a novel signal-on fluorimetric method for detecting OLP in both its pure form and aqueous humor. The proposed approach depends on enhancing the weak intrinsic fluorescence emission of OLP, achieving a remarkable increase of up to 680% compared to its intrinsic fluorescence. This enhancement is achieved by forming micelles around protonated OLP using an acetate buffer (pH 3.6) and incorporating a solution of sodium dodecyl sulfate (SDS) surfactant. A strong correlation (R = 0.9996) is observed between the concentration of OLP and fluorescence intensities ranging from 1.0 to 100.0 ng mL-1 with a limit of detection of 0.22 ng mL-1. This described method is successfully employed for quantifying OLP in both its powder form and pharmaceutical eye drops. Furthermore, it demonstrates robust performance in determining OLP in artificial aqueous humor with a percentage recovery of 99.05 ± 1.51, with minimal interference from matrix interferents. Moreover, the greenness of the described method was evaluated.

Endothelin-1 Plasma and Aqueous Humor Levels in Different Types of Glaucoma: A Systematic Review and Meta-Analysis.

Background and Objectives: Several studies suggest the complex relationship between Endothelin-1 (ET-1) levels with various types of glaucoma. This systematic review and meta-analysis explore ET-1 levels in plasma and aqueous humor among different types of glaucoma. Materials and Methods: A literature search (PubMed, ScienceDirect, Cochrane Library) was made up to April 2024 (PROSPERO: CRD42023430471). The results were synthesized according to PRISMA Guidelines. Results were presented as standardized mean differences (SMD) with 95% confidence intervals (CI). Results: A total of 2597 subjects (1513 patients with glaucoma vs. 1084 healthy controls) from 23 studies were included in a meta-analysis. Notably, patients with glaucoma reported significantly higher plasma levels of ET-1 compared to controls (SMD: 1.21, 95% CI: 0.59-1.82, p < 0.001). Particularly, plasma ET-1 levels were higher in primary open-angle glaucoma (POAG) (SMD: 0.87, 95% CI: 0.09-1.65, p < 0.05), normal-tension glaucoma (SMD: 0.86, 95% CI: 0.27-1.46, p = 0.05), and angle-closure glaucoma patients (SMD: 1.03, 95% CI: 0.43-1.63, p < 0.001) compared to healthy controls. Moreover, ET-1 aqueous humor levels were significantly higher in patients with glaucoma compared to controls (SMD: 1.60, 95% CI: 1.04-2.15, p < 0.001). In particular, aqueous humor levels were higher in POAG patients (SMD: 2.03 95% CI: 1.00-3.14, p < 0.001), and pseudoexfoliative glaucoma patients (SMD: 2.03, 95% CI: 1.00-3.07, p < 0.001) compared to controls. Conclusions: This meta-analysis indicates that elevated levels of ET-1 plasma and aqueous humor are significantly associated with different types of glaucoma. The pathogenesis of ET-1-related mechanisms may vary across different glaucoma types, indicating that possible therapeutic approaches targeting ET-1 pathways should be tailored to each specific glaucoma type.

Proteomics Analyses of Small Extracellular Vesicles of Aqueous Humor: Identification and Validation of GAS6 and SPP1 as Glaucoma Markers.

Cataracts and glaucoma account for a high percentage of vision loss and blindness worldwide. Small extracellular vesicles (sEVs) are released into different body fluids, including the eye's aqueous humor. Information about their proteome content and characterization in ocular pathologies is not yet well established. In this study, aqueous humor sEVs from healthy individuals, cataracts, and glaucoma patients were studied, and their specific protein profiles were characterized. Moreover, the potential of identified proteins as diagnostic glaucoma biomarkers was evaluated. The protein content of sEVs from patients' aqueous humor with cataracts and glaucoma compared to healthy individuals was analyzed by quantitative proteomics. Validation was performed by western blot (WB) and ELISA. A total of 828 peptides and 192 proteins were identified and quantified. After data analysis with the R program, 8 significantly dysregulated proteins from aqueous humor sEVs in cataracts and 16 in glaucoma showed an expression ratio ≥ 1.5. By WB and ELISA using directly aqueous humor samples, the dysregulation of 9 proteins was mostly confirmed. Importantly, GAS6 and SPP1 showed high diagnostic ability of glaucoma, which in combination allowed for discriminating glaucoma patients from control individuals with an area under the curve of 76.1% and a sensitivity of 65.6% and a specificity of 87.7%.

Analysis of Sphingosine and Sphinganine from the Aqueous Humor for Signaling Studies Using Ultrahigh-Performance Liquid Chromatography-Mass Spectrometry.

Sphingolipids, including sphingosine and sphinganine, are one of the major classes of lipids. They serve as constituents of cell membranes and lipid rafts and aid in the performance of cell-cell communication and adhesion. Abnormal levels of sphingolipids in the aqueous humor can indicate impaired sphingolipid metabolism and associated ocular pathologies. Sphingolipids can be extracted from the aqueous humor by the methyl-tert-butyl ether (MTBE) lipid extraction method and subsequently analyzed by liquid chromatography-mass spectrometry (LC-MS). This chapter describes a modified protocol for an MTBE lipid extraction from the aqueous humor, followed by analysis with ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS).

Parallel Analysis of Exosomes and Cytokines in Aqueous Humor Samples to Evaluate Biomarkers for Glaucoma.

Recent emerging studies have demonstrated numerous critical roles of exosomes in cell-to-cell signaling. We investigated exosomes in the aqueous humor of glaucoma patients and controls and compared their characteristics with other biomarkers such as cytokines. Glaucoma patients exhibited higher exosome particle counts and smaller sizes compared to controls. Higher exosome density was correlated with more severe visual field loss. Conversely, concentrations of aqueous humor cytokines, particularly PD-L1, were primarily associated with intraocular pressure, and none of the cytokines showed a significant association with visual field damage. This may reflect the characteristics of exosomes, which are advantageous for crossing various biological barriers. Exosomes may contain more information about glaucoma functional damage occurring in the retina or optic nerve head. This highlights the potential importance of exosomes as signaling mediators distinct from other existing molecules.

Stability of ascorbic acid in human aqueous humor.

OBJECTIVE: The stability of ascorbic acid (AA) in the human aqueous humor (AqH) remains unclear. This study aimed to investigate the stability of AqH AA under varying conditions (27, 4, - 20, and - 80 °C) without acidification. RESULTS: Rapid AA degradation occurred at 27 °C. At 4 °C, a significant 12.2% degradation was observed after 24 h. Storage at - 20 °C resulted in a notable 37.5% degradation after 28 days, whereas storage at - 80 °C resulted in 10.7% degradation after 28 days. Unacidified AqH samples recorded early decomposition at 27 °C and 4 °C. In conclusion, it is recommended to conduct measurements within 28 days for samples stored at - 80 °C.

Implications of Ocular Confounding Factors for Aqueous Humor Proteomic and Metabolomic Analyses in Retinal Diseases.

Purpose: To assess the impact of ocular confounding factors on aqueous humor (AH) proteomic and metabolomic analyses for retinal disease characterization. Methods: This study recruited 138 subjects (eyes): 102 with neovascular age-related macular degeneration (nAMD), 18 with diabetic macular edema (DME), and 18 with cataract (control group). AH samples underwent analysis using Olink Target 96 proteomics and Metabolon's metabolomics platform Data analysis included correlation, differential abundance, and gene-set analysis. Results: In total, 756 proteins and 408 metabolites were quantified in AH. Total AH protein concentration was notably higher in nAMD (3.2-fold) and DME (4.1-fold) compared to controls. Pseudophakic eyes showed higher total AH protein concentrations than phakic eyes (e.g., 1.6-fold in nAMD) and a specific protein signature indicative of matrix remodeling. Unexpectedly, pupil-dilating drugs containing phenylephrine/tropicamide increased several AH proteins, notably interleukin-6 (5.4-fold in nAMD). Correcting for these factors revealed functionally relevant protein correlation clusters and disease-relevant, differentially abundant proteins across the groups. Metabolomics analysis, for which the relevance of confounder adjustment was less apparent, suggested insufficiently controlled diabetes and chronic hyperglycemia in the DME group. Conclusions: AH protein concentration, pseudophakia, and pupil dilation with phenylephrine/tropicamide are important confounding factors for AH protein analyses. When these factors are considered, AH analyses can more clearly reveal disease-relevant factors. Translational Relevance: Considering AH protein concentration, lens status, and phenylephrine/tropicamide administration as confounders is crucial for accurate interpretation of AH protein data.

Impact of pH modification of the empirically used tobramycin ophthalmic solution on MIC90 concentration in tears and aqueous humor of donkeys (Equus asinus).

BACKGROUND: Commercial tobramycin ophthalmic solution is frequently used empirically to treat ocular disorders in equines, despite being primarily formulated for use in humans. It has been noted that tobramycin MIC90 concentration (minimal inhibitory concentration to 90% of microbial growth) rapidly declined following topical administration. It is hypothesized that adjustment of the pH of the empirically used tobramycin ophthalmic solution -prepared for human use- with the pH of the tears of donkeys, could increase the bioavailability of the drug and subsequently improve its penetration to the aqueous humor. Therefore, this study aimed to evaluate the impact of pH adjustment of the empirically used tobramycin ophthalmic solution on MIC90 concentration in tears and aqueous humor of donkeys (Equus asinus). The study was conducted on six (n = 6) clinically healthy donkeys. In each donkey, one eye was randomly selected to receive 210 µg tobramycin of the commercial tobramycin (CT) and used as a positive control (C group, n = 6). The other eye (treated eye) received 210 µg of the modified tobramycin ophthalmic solution (MT) (T group, n = 6). Tears and aqueous humor samples were collected 5-, 10-, 15-, 30- min, and 1-, 2-, 4-, and 6 h post-instillation. RESULTS: Modifying the pH of the empirically used commercial tobramycin ophthalmic solution in donkeys at a pH of 8.26 enhanced the drug's bioavailability. The MIC90 of the most hazardous bacteria isolated from equines' eyes such as Pseudomonas aeruginosa (MIC90 = 128 µg/ml) and Staphylococcus aureus (MIC90 = 256 µg/ml) was covered early (5 min post-instillation) and over a longer period in donkey tears (239-342 min) and aqueous humor (238-330 min) with the modified tobramycin solution. CONCLUSIONS: Adjustment of the pH of the commercial tobramycin ophthalmic solution, empirically used by veterinarians to treat donkeys' ophthalmic infections at a pH of 8.26, isotonic with the donkeys' tears pH, resulting in higher concentrations of tobramycin in tears and aqueous humor for a longer time.

Comparative analysis of human tear fluid and aqueous humor proteomes.

PURPOSE: Technological advancements allowing for the analysis of low-volume samples have led to the investigation of human tear fluid and aqueous humor (AH) as potential biomarker sources. However, acquiring AH samples poses significant challenges, making human tear fluid a more accessible alternative. This study aims to compare the protein compositions of these two biofluids to evaluate their suitability for biomarker discovery. METHODS: Paired tear and AH samples were collected from 20 patients undergoing cataract surgery. Tear samples were collected using Schirmer strips prior to surgery, and AH samples were collected from the anterior chamber immediately after corneal incision. Proteins were extracted and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: A total of 481 proteins were identified in greater than 50% of the tear samples, and 191 proteins were detected in greater than 50% of the AH samples. Of these proteins, 82 were found to be common between the two biofluids, with ALB, LTF, TF, LCN1, and IGKC being the most abundant. CONCLUSION: Although tear fluid and the AH are functionally independent and physically separated, many of the proteins detected in AH were also detected in tears. This direct comparison of the proteomic content of tear fluid and AH may aid in further investigation of tear fluid as a source of readily accessible biomarkers for various human diseases.

A novel biomarker assay qualification strategy for rare human matrices - a case study of biomarkers in aqueous humor.

Background: Measuring pharmacodynamic biomarkers near the therapeutic site of action presents considerable challenges for sites with limited matrix volume or difficult access. Bioanalytical method qualification requires the use of numerous matrix samples, which is problematic for rare matrices. The aim of this study was to design and implement a streamlined, fit-for-purpose strategy for qualification of biomarker assays in rare matrices. Materials & methods: A multiplexed biomarker immunoassay was developed in human aqueous humor. Results: Our strategy was successfully implemented, providing characterization of assay performance while reducing number of samples in assay qualification. Our assay was used in clinical trial support for an ophthalmic drug candidate. Conclusion: Our results indicate this approach can be applied to other early stage drug development programs facing similar challenges.

Bioanalytical assay development and qualification requires the use of numerous matrix samples, which is problematic for rare matrices. We designed and successfully implemented a streamlined strategy for qualification of biomarker assays in rare matrices.

PCR testing for herpesviruses in aqueous humor samples from patients with and without clinical corneal endothelial graft rejection.

To compare prevalence of positive PCR tests for herpesviruses between patients with and without a history of clinical corneal endothelial allograft rejection (AGR). Retrospective cross-sectional study with two-group comparison. A total of 307 aqueous humor (AH) samples from 235 Patients and 244 eyes who underwent penetrating keratoplasty or Descemet membrane endothelial keratoplasty or had a diagnostic AH aspiration due to clinical AGR between 2019 and 2023 were tested for DNA of herpes simplex virus (HSV), varicella-zoster virus (VZV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV). PCR test results were compared between the two groups (with/without AGR). Another sub-analysis examined the results of patients without a history of herpetic keratitis. A total of 8% of eyes with clinical AGR (9/108) had a positive PCR result for one of the herpesviruses (HSV:3, CMV:3, EBV:2, VZV:1). All patients in the group without AGR had negative PCR results for all previous viruses (0/136). The difference was statistically significant (p < 0.001). The sub-analysis of eyes without a history of herpetic keratitis also revealed significantly more positive herpes PCR results (7/87) in eyes with AGR than in eyes without AGR (0/42, p = 0.005). Clinical AGR after keratoplasty shows a significant correlation to viral replication. Herpetic infection and AGR could occur simultaneously and act synergistically. Timely differentiation between active herpetic infection and/or AGR is pivotal for proper treatment and graft preservation.

Liquid-biopsy proteomics combined with AI identifies cellular drivers of eye aging and disease in vivo.

Single-cell analysis in living humans is essential for understanding disease mechanisms, but it is impractical in non-regenerative organs, such as the eye and brain, because tissue biopsies would cause serious damage. We resolve this problem by integrating proteomics of liquid biopsies with single-cell transcriptomics from all known ocular cell types to trace the cellular origin of 5,953 proteins detected in the aqueous humor. We identified hundreds of cell-specific protein markers, including for individual retinal cell types. Surprisingly, our results reveal that retinal degeneration occurs in Parkinson's disease, and the cells driving diabetic retinopathy switch with disease stage. Finally, we developed artificial intelligence (AI) models to assess individual cellular aging and found that many eye diseases not associated with chronological age undergo accelerated molecular aging of disease-specific cell types. Our approach, which can be applied to other organ systems, has the potential to transform molecular diagnostics and prognostics while uncovering new cellular disease and aging mechanisms.

Exquisite integration of quality-by-design and green analytical approaches for simultaneous determination of xylometazoline and antazoline in eye drops and rabbit aqueous humor, application to stability study.

This work implements a stability indicating HPLC method developed to simultaneously determine xylometazoline (XYLO) and antazoline (ANT) in their binary mixture, rabbit aqueous humor and cited drug's degradates by applying analytical quality-by-design (AQbD) combined with green analytical chemistry (GAC) experiment for the first time. This integration was designed to maximize efficiency and minimize environmental impacts, as well as energy and solvent consumption. Analytical quality-by-design was applied to achieve our aim starting with evaluation of quality risk and scouting analysis, tracked via five parameters chromatographic screening using Placket-Burman design namely: pH, temperature, organic solvent percentage, flow rate, and wavelength detection. Recognizing the critical method parameters was done followed by optimization employing central composite design and Derringer's desirability toward assess optimum conditions that attained best resolution with satisfactory peak symmetry with short run time. Optimal chromatographic separation was attained by means of an XBridge® C18 (4.6 × 250 mm, 5 µm) column through isocratic elution using a mobile phase consists of phosphate buffer (pH 3.0): ethanol (60:40, by volume) at a 1.6 mL/min flow rate and 230.0 nm UV detection. Linearity acquired over a concentration range of 1.0-100.0 µg/mL and 0.5-100.0 µg/mL for XYLO and ANT, respectively. Furthermore, imperiling cited drugs' stock solutions to stress various conditions and satisfactory peaks of degradation products were obtained indicating that cited drugs are vulnerable to oxidative degradation and basic hydrolysis. Degradates' structures were elucidated using mass spectrometry. Applying various assessment tools; namely: analytical greenness (AGREE), green analytical procedure index (GAPI), analytical eco-scale, and national environmental method index (NEMI), Greenness method's evaluation was applied and proved to be green. In fact, the developed method is established to be perceptive, accurate, and selective to assess cited drugs for routine analysis.

Development and Validation of Facile RP-HPLC Method for Simultaneous Determination of Timolol Maleate, Moxifloxacin Hydrochloride, Diclofenac Sodium and Dexamethasone in Plasma, Aqueous Humor and Pharmaceutical Products.

The present study aimed to develop a validated RP-HPLC method for the simultaneous determination of timolol maleate (TM), moxifloxacin hydrochloride (MOXI), diclofenac sodium (DS) and dexamethasone (DEXA) in human plasma, bovine aqueous humor and pharmaceutical preparations. The chromatographic separation was studied using the C18 column. The chromatographic conditions, such as composition, pH, the flow rate of mobile phase, the temperature of column, wavelength of absorption and injection volume of the sample, were studied. The method was validated to confirm specificity, linearity and accuracy in accordance with an International Conference on Harmonization guidelines. The optimum conditions for separation included mobile phase 0.05 M monobasic phosphate buffer: acetonitrile (65:35 v/v), pH of buffer adjusted to 6.2 and the flow rate of 1 mL/minute. The optimum temperature of the column was found to be 35°C, absorption wavelength 265 nm and injection volume 50 µL. The baseline separation of all four drugs with good sensitivity, resolution, and a less than 15 min run time was achieved. The retention time of TM, MOXI, DS and DEXA were 4.3,5.7,9.9 and 13.5 minutes respectively. The limit of detection (LOD) values were 6.2, 4.8, 0.8 and 1.2 ng/mL for TM, MOXI, DS and DEXA, respectively, whereas their respective limit of quantification (LOQ) values was: were 42.6, 26.8, 5.6 and 6.2 ng/mL. The coefficient of variation for intra-day and inter-day were in the range of 0.32-1.57 and 1.29-3.07%, respectively. The method was found to be sensitive, precise and accurate in human plasma and bovine aqueous humor and can be applied for the quantification of these compounds in plasma, aqueous humor and pharmaceuticals.

Chromatographic Analysis of Aqueous Humor of Bupivacaine in Different Administration Approaches.

Different administration approaches were investigated for the selection of bupivacaine administration type and a sensitive high-performance liquid chromatographic (HPLC) method has been developed. Developed method was validated and applied for the determination of bupivacaine in rabbit aqueous humor. The separation was achieved using a XTerra, C8 (250 × 8 mm i.d., particle size 5 µm) analytical column with a mobile phase consisted of acetonitrile and sodium dihydrogen phosphate (pH = 3.0, 20 mM; 30:70, v/v). Bupivacaine detection was performed by Diode Array detector (DAD) at 220 nm. The retention times for bupivacaine is 15.886 min. HPLC-DAD method was linear in the range of 75-4000 ng/mL. The limit of detection was 25 ng/mL and the limit of quantification of bupivacaine was found to be 75 ng/mL (relative standard deviation, RSD ≤ 15%, n = 6). In intra-day and inter-day precision and accuracy analysis, the RSD was found to be in the range of 0.96 and 7.98%, the bias values were 0.64 and 3.33%. Method was carried out for three different type of bupivacaine application because of the investigation of effective drug administration. Twenty aqueous humor samples were in the range of 0.642 and 5.124 µg/mL.

Metabolomics Analysis of Aqueous Humor Based on Liquid Chromatography-Mass Spectrometry.

Aqueous humor (AH) is a transparent fluid that fills the anterior segment of the eye. The composition and level of metabolites in AH are important for understanding its physiology and changes caused by the occurrence of eye disease. A simple method for the preparation and analysis of AH samples was developed using the liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) technique. The analyses were performed using two types of chromatography: reversed-phase liquid chromatography-mass spectrometry (LC-RP-MS) and hydrophilic interaction liquid chromatography-mass spectrometry (LC-HILIC-MS), in the sample prepared by one protocol.